 |
INTRODUCTION |
Mammalian adenylyl cyclases are a diverse group of variously
regulated signaling molecules. Details are emerging on some of the
molecular features conferring catalytic and regulatory properties on
these enzymes. All of the nine cloned adenylyl cyclases are large
(1080-1248 amino acids) polypeptides that are proposed to comprise two
cassettes of six transmembrane-spanning domains, each cassette being
followed by a large cytoplasmic domain (1, 2). The transmembrane
domains are not highly conserved among adenylyl cyclases. However,
parts of two of the cytoplasmic domains (termed C1a and C2a) are highly
conserved, and, when expressed separately, they can combine to display
basic catalytic activity (3-8). These molecules have been
crystallized, and the combination of C1a and C2a, each consisting of a
three layer
/
sandwich, forms an active catalytic core to
generate cAMP from ATP (9, 10). The other major cytoplasmic domains of
mammalian adenylyl cyclases, the N terminus, the C1b region, and the
C2b region, are not conserved at all and are speculated to reflect
regulatory features of specific adenylyl cyclases (1, 11-13).
Ca2+ elicits a prominent stimulation of
ACI1 and ACVIII, which is
mediated by loosely bound calmodulin (3, 11, 14). Although the likely
calmodulin-binding domain on ACI has been localized to the C1b region
(15, 16), the corresponding regulatory domain has not been identified
on ACVIII. Indeed, ACVIII does not possess analogous calmodulin-binding
sites in the C1b region. In the case of ACI, peptides corresponding to
putative calmodulin-binding domains were used to identify a site in the
C1b region as the likely site of calmodulin binding (16). Mutagenesis
studies strongly supported this assignment (15, 17). However, no
information is yet available on the possible domains that mediate the
Ca2+/calmodulin responsiveness of ACVIII. Given that ACI
and ACVIII do not share similar C1b domains (they are only ~40%
similar at the amino acid level, compared with 80% similarity in the
C1a region) and also that their regulation by
Ca2+/calmodulin shows distinct properties, it might not be
unexpected if different motifs and/or locations were involved.
Identifying calmodulin-binding sites on proteins still mainly depends
on experimentation, although some predictive criteria are available to
guide experiments. For instance, many known
Ca2+-dependent calmodulin-binding proteins
possess a region that is often characterized by an amphipathic helix
consisting of approximately 20 amino acid residues (18). In these
regions, basic amino acids are interspersed among hydrophobic residues,
and aromatic amino acids normally appear near either end (16, 18, 19).
However, sequence analysis based on these criteria does not always
identify calmodulin-binding regions, and indeed, regions of proteins
that bind to calmodulin sometimes do not fit these criteria. Another calmodulin-binding motif is the so-called "IQ motif," consensus sequence IQXXXRGXXXR, which often (18) but not
always (22, 23) binds calmodulin in a Ca2+-independent manner.
The present studies used a combination of calmodulin overlay assays,
mutagenesis, and peptide inhibition studies to locate the calmodulin
regulatory domains on ACVIII. Surprisingly, a primary site was located
in the C2b region, while an ancillary site that appeared to play a
minor autoinhibitory role was located in the N terminus.
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EXPERIMENTAL PROCEDURES |
Materials--
Thapsigargin, forskolin, and Ro 20-1724 were
from Calbiochem. [2-3H]Adenine, [3H]cAMP,
and [
-32P]ATP were obtained from Amersham Pharmacia
Biotech. The restriction enzymes and other enzymes used in subcloning
were from New England Biolabs (Beverly, MA). Other reagents were from
Sigma unless stated otherwise.
Production of His-tagged Protein Fragments from
ACVIII--
Eight His-tagged fusion proteins were generated for
calmodulin overlay experiments and are referred to as follows, with the appropriate ACVIII residues in parentheses: Nt
(Met1-Glu179); Nn
(Met1-Ser110); Nc
(Glu108-Glu179); C1
(Ala346-Asn712); C1a
(Ala346-Ser593); C2
(Gly913-Pro1248); C2a
(Gly913-Pro1184); C2b
(Leu1137-Pro1248). Nt, Nn, C2, C2a, and C2b
were constructed by amplifying nucleotides 777-1314, 777-1136,
3513-4520, 3513-4329, and 4185-4520, respectively, by polymerase
chain reaction (PCR) and subcloning the PCR products between
EcoRI (5'-end) and HindIII (3'-end, blunted)
sites of pRSETb (Invitrogen, Carlsbad, CA). C1 and C1a were constructed by amplifying nucleotides 1811-2912 and 1811-2546 by PCR and
subcloning the PCR products between NcoI (5'-end) and
HindIII (3'-end, blunted) sites of pRSETb. Nc was created
from the Nt construct by cutting with EcoRI and BspI,
blunting the two ends and ligating them back. Constructs were confirmed
by sequencing. These cDNA constructs were transformed into BL21
(DE3) pLys S cells. The cells were grown in Luria's broth containing
100 µg/ml ampicillin and 34 µg/ml chloramphenicol at 37 °C until
they reached an A600 of 0.8. Expression of these
proteins was induced by the addition of 0.5 mM isopropyl
-D-thiogalactoside and incubation of the cells overnight at 37 °C. The cells were harvested by centrifugation at 6000 × g for 10 min and resuspended in ice-cold extraction buffer
(20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1% Triton
X-100, 1 mM
-mercaptoethanol, and protease inhibitor
mixture). The cell lysate was sonicated (4 × 10 s) and
separated by centrifugation at 10,000 × g for 10 min.
The pellets were resuspended in equal volumes of 2× sample buffer,
boiled, and spun at 13,000 × g for 5 min to eliminate insoluble material. The sample buffer containing bacterial proteins was
loaded onto SDS-polyacrylamide gel electrophoresis gel. The expressed
fusion proteins could be visualized readily from the gel stained by
Coomassie Blue. These protein bands were also confirmed by Western blot
probed with RGS-His antibody (Qiagen, Valencia, CA).
Calmodulin Overlay Assay--
The calmodulin overlay assay was
performed by fractionating His-tagged fusion proteins by
SDS-polyacrylamide gel electrophoresis, transferring to nitrocellulose
membranes, and probing with biotinylated calmodulin, as described by
the manufacturer (Calbiochem). The blot membranes were stained with
India ink.
Mutagenesis--
Deletion mutants of ACVIII were generated
by using different strategies. Mutants C1
588-619,
C1
620-644, C1
588-644, and the
Phe158-Arg169 deletion of N
1-106,
158-169 were constructed by a PCR-based strategy (24). The
deleted ACVIII residues of the mutants above are shown in Table I.
Mutants N
1-106 and N
1-49 were
constructed by cutting pcDNA3/ACVIII plasmid with BspI
and EcoR47, respectively, blunting the ends with T4 DNA
polymerase, cutting again with NotI, and subcloning the
fragments into the EcoRI (blunted with T4 DNA polymerase) and NotI sites of pcDNA3/Hisb (Invitrogen, Carlsbad, CA)
in frame. C2
1126-1248 was generated by cutting
pcDNA3/ACVIII plasmid with BamHI and NotI,
polishing the ends with T4 DNA polymerase, and religating them back
together. Mutant C2
1184-1248 was constructed by cutting
the C2a fragment in pRSET with BamHI and BglII
(blunt) and subcloning into the BamHI and
NotI (blunt) sites of pcDNA3/ACVIII. Deletions
NC1
1-106,
635-700, NC1
1-106,
588-619, and
NC1
1-106,
620-644 were constructed by cutting
C1
635-700, C1
588-619, and
C1
620-644 with EcoRV and SfiI and
subcloning the fragments into the EcoRV and
SfiI sites of N
1-106 plasmid. Mutants
NC2
1-106,
1184-1248,
NC2
1-106,
158-169,
1184-1248, and
NC1C2
1-106,
635-700,
1184-1248 were
constructed by cutting N
1-106,
N
1-106,
158-169, and
NC1
1-106,
635-700 with EcoRV and
ApaI and subcloning the fragments into EcoRV and
ApaI sites of mutant C2
1184-1248.
All of the mutated cDNA constructs were confirmed by sequencing.
Measurement of cAMP Accumulation--
HEK 293 cells were
maintained and transfected as described previously (25). cAMP
accumulation in intact cells expressing different ACVIII mutant
constructs was measured according to the method of Evans et
al. (1984) as described previously (26, 27) with some
modifications. HEK 293 cells on 12-well plates were incubated in
minimal essential medium (60 min, 37 °C) with
[2-3H]adenine (1.5 µCi/well) to label the ATP pool. The
cells were then washed twice and incubated with a nominally
Ca2+-free Krebs buffer (900 µl/well) containing 120 mM NaCl, 4.75 mM KCl, 1.44 mM
MgSO4, 11 mM glucose, 25 mM HEPES,
and 0.1% bovine serum albumin (fraction V) adjusted to pH 7.4 with 2 M Tris base. The use of Ca2+-free Krebs buffer
in experiments denotes the addition of 0.1 mM EGTA to the
nominally Ca2+-free Krebs buffer. All experiments were
carried out at 37 °C in the presence of the phosphodiesterase
inhibitors, 3-isobutyl-1-methylxanthine (500 µM) and Ro
20-1724 (100 µM), which were preincubated with the cells
for 10 min prior to a 1-min assay. Unless stated otherwise, cells were
preincubated for 10 min with the Ca2+-ATPase inhibitor
thapsigargin at a final concentration of 100 nM. This has
the effect of passively emptying the Ca2+ stores,
establishing a low basal [Ca2+]i, and priming the
cells for capacitative Ca2+ entry (28). Assays were
terminated by the addition of 5% (w/v final concentration)
trichloroacetic acid. Unlabeled cAMP (100 µl, 10 mM), ATP
(10 µl, 65 mM), and [
-32P]ATP(~7000
cpm) were added to monitor recovery of cAMP and ATP. After pelleting,
the [3H]ATP and [3H]cAMP content of the
supernatant were quantified according to the standard Dowex/alumina
methodology (29). The accumulation of cAMP is expressed as the
percentage of conversion of [3H]ATP into
[3H]cAMP; means ± S.D. of triplicate determinations
are indicated. The basal activity of some constructs was high, with the
result that significant amounts of cAMP had accumulated before the
beginning of the 1-min incubation period. In such cases, the Krebs
buffer bathing the cells was exchanged with buffer containing the same concentrations of 3-isobutyl-1-methylxanthine/Ro20-1724, EGTA, and
thapsigargin, at the beginning of the 1-min assay, as indicated in the
figure legends.
Preparation of Plasma Membranes from Transfected HEK 293 Cells--
HEK 293 cells, transfected with pcDNA3 vector alone,
ACVIII, or ACVIII mutant plasmids, were lysed using a modification of a
glycerol-stabilized lysis method (30, 31). The lysate from six
75-cm2 flasks was adjusted to 3% sucrose, layered on top
of linear sucrose gradients (5-45%), and centrifuged at 34,000 rpm (1 h, 4 °C, SW40 rotor, Beckman Instruments, Palo Alto, CA). The plasma
membranes appeared as a clear band around 32-38% sucrose as described
previously (32). The plasma membrane protein band was pelleted in 20 mM HEPES (pH 7.4) buffer and resuspended in the assay
buffer (200 mM Tris pH 7.4, 800 µM EGTA, and
0.25% bovine serum albumin (fraction V)).
Western Blotting Experiments--
Two antibodies were used in
Western blot experiments. Ab VIII-A 1229-1248 was raised against amino
acids 1229-1248 of ACVIII at the very end of the C terminus; the
other, Ab VIII-A 666-682, was raised against amino acids 666-682 in
the C1b region of ACVIII (33). The experimental procedure was described
previously (33). 10-µg membrane proteins were loaded on each lane of
a 10% polyacrylamide gel. After electrophoresis, the contents of the
polyacrylamide gel were transferred to polyvinylidene difluoride
membranes (Micron Separations Inc.), which were incubated with TBST (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.1% Tween
20) containing 5% nonfat dry milk, for 1 h at room temperature.
The primary antibody (1:20,000 dilution) was then added, and the
membrane was incubated for another 1 h. After washing in TBST, the
membrane was incubated in TBST containing 5% nonfat dry milk with goat
anti-rabbit IgG horseradish peroxidase conjugate (1:1000 dilution;
Bio-Rad) at room temperature for 1 h. The immune complex was
detected by enhanced chemiluminescence (manufacturer's protocol;
Amersham Pharmacia Biotech).
Adenylyl Cyclase Activity Measurements--
Determination of
adenylyl cyclase activity in vitro was performed as
described previously (34) on isolated transfected HEK 293 cell
membranes. The adenylyl cyclase activity of the HEK 293 cells was
measured in the presence of the following components: 12 mM
phosphocreatine, 2.5 units of creatine phosphokinase, 0.1 mM cAMP, 1 mM MgCl2, 0.1 mM ATP, 70 mM Tris buffer, pH 7.4, 0.04 mM GTP, 1 µCi of [
-32P]ATP, calmodulin
(1 µM), and forskolin (20 µM), as
indicated. Free Ca2+ concentrations were established from a
series of CaCl2 solutions buffered with 200 µM EGTA in the assay (34). The reaction mixture (final
volume, 100 µl) was incubated at 30 °C for 20 min. Reactions were
terminated with sodium lauryl sulfate (0.5%); [3H]cAMP
was added as a recovery marker, and the [32P]cAMP formed
was quantitated as described previously (29). Data points are presented
as mean activities ± S.D. of triplicate determinations.
Protein Secondary Structure Prediction and Synthetic Peptide
Experiments--
The protein sequence was analyzed with DNASIS 2.5 (Hitachi Software Engineering Co. Ltd., San Bruno, CA). The secondary
structures of the N-terminal fragment and the C2b region were predicted
using the Chou and Fasman program. Two peptides were synthesized
(Genemed Synthesis Inc., South San Francisco, CA). One was 21 residues, called 8Ncam (RPQRLLWQTAVRHITEQRFIH), corresponding to amino acids 32-52 of ACVIII; the other one was 25 residues, called 8Ccam
(YSLAAVVLGLVQSLNRQRQKQLLNE), corresponding to amino acids 1186-1211 of
ACVIII. The N termini of the two peptides were protected by
acetylation, and the C termini were protected by amidation, to prevent
self-circulation and to mimic the in vivo conformation. Both
peptides were purified above 95%. The molecular weight for 8Ncam and
8Ccam is 2686 and 2885, respectively. The purity and molecular weight
of the peptides were confirmed by high pressure liquid chromatography
and mass spectroscopy, respectively (Genemed Synthesis). The peptide
(CamkII, LKKFQARRKLKGAILTTMLA) of the CAM kinase II calmodulin-binding domain was purchased from Calbiochem. The peptide 8CT
(TPSGPEPGAQAEGTDKSDLP) has 20 residues, corresponding to amino acids
1229-1248 of ACVIII, which was originally synthesized for raising
antibody Ab VIII-A 1229-1248 (33). Stock solutions (2 mM)
were made in water for all four peptides, which were used in in
vitro adenylyl cyclase assays as described above, to generate
competition and inhibition curves. The inhibition curves were fitted by
the program Inplot 4.04 (GraphPad Software Inc.) with the following
equation: Y = A + (B
A)/(1 + (X/C)), where Y
represents the adenylyl cyclase activity relative to the activity in
the absence of peptides and X is the peptide concentration.
The maximal value (A), the minimal value (B), and
IC50 (C) were obtained by curve fitting. The
IC50 for each peptide was calculated from three independent experiments.
 |
RESULTS |
Calmodulin Overlay Assays--
Overlay assays were an initial
strategy adopted to identify putative regions for calmodulin regulation
of ACVIII. It seemed reasonable to assume that a sizable molecule
(~19 kDa) such as calmodulin, which is dissociable from ACVIII by
washing in EGTA-containing buffers (14), should bind somewhere in the
three large intracellular loops. Consequently, eight fusion proteins
including the regions comprising these three loops were generated,
designated Nt, Nn, Nc, C1, C1a, C2, C2a, and C2b (Fig.
1A) as described under
"Experimental Procedures." Western blotting with an anti-RGS-His
antibody indicated that most of the expressed fusion proteins were in
the pellets from the Escherichia coli cell lysates (data not
shown). Therefore, cell lysate pellets solubilized with sample buffer
were used in calmodulin overlay assays. Following transfer from
SDS-polyacrylamide gel electrophoresis, nitrocellulose membranes with
fractionated E. coli proteins were incubated with
biotinylated calmodulin, and the bands that bound calmodulin were
detected by horseradish peroxidase-streptavidin incubation and enhanced
chemiluminescence (see "Experimental Procedures"). The N-terminal
fragments, Nt and Nn, yielded strong signals for calmodulin binding
(Fig. 1, C and E). This signal was
Ca2+-dependent and was removable by washing the
membrane with EGTA-containing buffer (data not shown). No significant
signal was detected for any other domains (Fig. 1, C and
E). Two other fusion proteins including the C1b region were
also generated, neither of which showed positive results in calmodulin
overlay assays (data not shown). Thus, these data indicate that the
N-terminal exclusively binds calmodulin. However, since proteins on
nitrocellulose membranes are denatured, it is quite possible that a
site binding to calmodulin in the native state would not bind
calmodulin in an overlay assay. Therefore, mutagenesis studies and
functional experiments were considered critical to evaluate the
significance of any apparent interactions derived from overlay
assays.

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Fig. 1.
Calmodulin overlay assays. A,
diagram of protein fragments from ACVIII cytoplasmic domains. Five
amino acids and their positions are marked in the one-dimensional
structure diagram of ACVIII from left (N terminus) to right (C
terminus). Vertical black boxes
represent the predicted transmembrane domains; two
thick lines represent the highly conserved C1a
and the C2a region. The positions of eight cytoplasmic fragments
(horizontal black boxes) are shown
below, which were generated into His-tagged fusion proteins
as described under "Experimental Procedures." B-E,
calmodulin overlay assays were performed as described under
"Experimental Procedures" in the presence of 0.5 mM
CaCl2. The fusion proteins loaded on the gel are indicated
at the top of the panels. The molecular weight is
marked at the left. B and D,
transferred membrane blots stained with India ink. C and
E, calmodulin binding shown in the x-ray film from the
membranes of B and D, respectively. The positions
of the fusion proteins are indicated (arrowheads), which
were confirmed by Western blot probed with anti-RGS His antibody.
Nonspecific binding to horseradish peroxidase-streptavidin is also
indicated (asterisk). When 0.5 mM EGTA was
substituted for CaCl2 in the overlay assay, the
calmodulin-binding bands in C and E were no
longer evident (not shown).
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Ca2+ Sensitivity of ACVIII Mutants in Vivo--
The
C1a and C2a regions, which are the catalytic domains (4, 6, 35) are
highly conserved across the mammalian adenylyl cyclase family. These
domains have already been crystallized and well characterized (5, 9,
10, 36, 37). A putative calmodulin-binding site was not revealed in
these highly conserved regions by either sequence analysis or earlier
experimental attempts (15, 16). On the other hand, the N terminus, C1b,
and C2b regions are poorly conserved and are the regions where
type-specific regulatory domains would be expected to reside.
Therefore, a group of deletions was made, concentrating on these three
areas (Table I). These mutant constructs
were expressed in HEK 293 cells by transient transfection, and 48 h later, cAMP accumulation was measured in the intact HEK 293 cells in
response to capacitative Ca2+ entry. The cells were
pretreated with 100 nM thapsigargin to deplete the
intracellular Ca2+ stores and prime the cells for
capacitative Ca2+ entry, which selectively regulates the
activities of Ca2+-sensitive adenylyl cyclases (25, 38,
39). Thus, the Ca2+ sensitivity of adenylyl cyclases can be
determined by comparing the cAMP accumulation in cells pretreated with
thapsigargin in the presence or absence of Ca2+ during the
assay. Surprisingly, the N terminus deletion of ACVIII, N
1-106 (mutant 1 in Fig.
2A) remained sensitive to Ca2+, although this deleted region bound calmodulin in the
overlay assay (Fig. 1). The -fold stimulation by Ca2+ for
N
1-106 and two other N terminus deletions,
N
1-49 (Fig. 2A, mutant 2) and
N
1-106,
158-169 (mutant 3) was only about half of
that for wild type ACVIII; nevertheless, clear Ca2+
regulation was retained. These data strongly suggested that the N-terminal region was not the only functionally significant
calmodulin-binding region within ACVIII.

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Fig. 2.
The sensitivity of ACVIII mutants to
capacitative Ca2+ entry. Assays (1 min) were performed
in intact, transfected cells pretreated with
3-isobutyl-1-methylxanthine/Ro20-1724, EGTA and thapsigargin, as
described under "Experimental Procedures." A, the effect
of capacitative Ca2+ entry on cAMP accumulation in cells
transfected with vector alone (pcDNA3.0), wild type (AC8), and
mutants. Mutant numbers as identified in Table I are used. cAMP
accumulation was measured under three assay conditions: basal
(including 0.2% Me2SO; black bars);
forskolin (20 µM; white bars); and
forskolin (20 µM) plus external CaCl2
(4 mM; gray bars). The data shown are
representative of at least three similar experiments. B, a
modified in vivo assay protocol was used as described under
"Experimental Procedures" to diminish the influence of
extracellular cAMP accumulation in the preincubation. Cells transfected
with vector alone (pcDNA3.0), ACVIII wild type,
C2 1184-1248, and
NC2 1-106, 1184-1248, were assayed. 10%
trichloroacetic acid was added at the beginning of a 1-min assay
to one group of transfected cells to obtain a
T0 value. This value (0.1% for vector; 0.5%
for ACVIII; 1.34% for C2 1184-1248; 7.8% for
NC2 1-106, 1184-1248) was subtracted from the
subsequent values, in which cAMP accumulation was measured in the
indicated groups for 1 min at basal condition (dark
gray bars), 4 mM external
CaCl2 (white bars), 20 µM forskolin (black bars), or 20 µM forskolin and 4 mM external
CaCl2 (light gray
bars), as indicated.
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Deletions in the C1b region were next evaluated. Deleting the entire
C1b region (Pro587-Ser701) fully inactivated
ACVIII (data not shown). The mutant C1
635-700 (Fig.
2A, mutant 4), which is a naturally occurring splice variant of ACVIII (the "C form" (33), which lacks 66 amino acids in the C1b
region), was stimulated by Ca2+. A deletion
C1
588-644 (Fig. 2A, mutant 7), missing the
remaining part of the C1b region, appeared to lose its sensitivity to Ca2+, but its activity was very low. Two related
deletions, C1
588-619 (Fig. 2A, mutant 5) and
C1
620-644 (mutant 6), were similar to the
wild type ACVIII in terms of their -fold stimulation by
Ca2+, although the forskolin- and
Ca2+-stimulated activity of C1
620-644 was
much higher than that of C1
588-619. These results
suggest that the C1b region is important for ACVIII activity, and,
consequently, deletions in the C1b region could modify the
Ca2+ regulation and the catalytic activity of ACVIII.
However, it seems unlikely that this region is involved in the direct
regulation by calmodulin. A double deletion,
N
1-106,
588-619 (Fig. 2A, mutant 10),
lacking approximately two-thirds of the N terminus and a major part of
the C1b region, was also Ca2+-stimulable. This mutant
further confirmed that the N terminus and the C1b region are unlikely
to be the calmodulin-binding region responsible for stimulation by
Ca2+.
Finally, the C2b region of ACVIII was explored. An extensive C2b
deletion, C2
1126-1248 (Fig. 2A, mutant 8)
was inactive. Another C2b mutant, C2
1184-1248 (mutant
9) showed extremely high basal activity, which was comparable
with wild type ACVIII activity when the latter was stimulated by
forskolin and Ca2+. Forskolin could further stimulate
C2
1184-1248 activity by about 50%, but
Ca2+ could evoke no stimulation (Fig. 2A). An
additional deletion of C2
1184-1248 in the N terminus,
NC2
1-106,
1184-1248 (Fig. 2A, mutant
13), resulted in an even higher basal cAMP accumulation. The high basal
cAMP accumulation of NC2
1-106,
1184-1248 coupled
with its insensitivity to stimulation suggests the removal of
inhibitory influences. Two other multiple deletions, including the C2b
region,
NC2
1-106,
158-169,
1184-1248 (Fig.
2A, mutant 14) and
NC1C2
1-106,
158-169,
636-702,
1184-1248 (mutant 15), were also insensitive to forskolin and Ca2+,
although the basal activity of both of these multiple deletions was
much lower than that of NC2
1-106,
1184-1248.
In the assays described above, the basal activity of adenylyl cyclase
is the cAMP that accumulates both inside and outside the cells before
(10 min) and during the 1-min assay, in the presence of PDE inhibitors
to prevent the breakdown of cAMP. Basal accumulation of cAMP is
normally trivial during the preincubation period; however, where basal
activities of some of these constructs are high (particularly C2b
deletions), a considerable accumulation of cAMP can occur prior to the
onset of the 1-min assay. Therefore, to clearly examine the activity of
NC2
1-106,
184-1248 within the 1-min assay period,
we modified our in vivo assay protocol. The medium was
exchanged with new buffer just before the 1-min assay to minimize the
influence of accumulated extracellular cAMP. In addition, the
intracellular cAMP that had accumulated was measured by
exchanging the medium and lysing one group of cells with
trichloroacetic acid at the beginning of the 1-min assay. When
HEK 293 cells transfected with pcDNA3, ACVIII,
C2
1184-1248, or
NC2
1-106,
1184-1248 were assayed using this
modified protocol, the cAMP accumulation of
C2
1184-1248 and
NC2
1-106,
1184-1248 in all of the
conditions was much reduced. However, stimulation by Ca2+
of wild type ACVIII was evident even in the basal state and was strikingly evident when activity was also stimulated by forskolin (Fig. 2B). A clear stimulation of
NC2
1-106,
1184-1248 by forskolin was also revealed
(Fig. 2B), while in the original in vivo assay no
significant forskolin stimulation was detectable, probably due to
masking by the high extracellular cAMP accumulation (Fig.
2A). Most significantly, again, in keeping with the original in vivo assay results (Fig. 2A), both
C2
1184-1248 and NC2
1-106,
1184-1248 were quite
insensitive to Ca2+ (Fig. 2B). Overall, these
data strongly indicate that the C2b region of ACVIII is critical to the
Ca2+ stimulation. The activity of these deletions was also
assessed in in vitro assays to confirm or disprove the
conclusions from the intact cell assays.
Ca2+ Sensitivity of ACVIII Mutants in
Vitro--
Plasma membranes were prepared from transfected HEK 293 cells, and adenylyl cyclase activity was assayed with either 20 µM forskolin or 20 µM forskolin plus 2 µM free Ca2+. All of the mutants in the N
terminus and the C1b regions were sensitive to Ca2+ except
C1
588-644, whose activity was close to the background
(Fig. 3). Mutants N
1-106
and C1
620-644 showed higher -fold stimulation by
Ca2+ than did the wild type (Fig. 3). Three double
deletions, NC1
1-106,
635-700, NC1
1-106,
588-619 (Fig. 3, mutant 11), and
NC1
1-106,
620-644 (mutant 12), could also still be
stimulated by Ca2+. As anticipated from the in
vivo studies, deletion of the C2b region
(C2
1184-1248), resulted in a loss of the
Ca2+ sensitivity (Fig. 3). The activity of the double
deletion, NC2
1-106,
1184-1248, was inhibited
(rather than stimulated) approximately 20% by 2 µM free
Ca2+ (Fig. 3). Although the activity of this latter
construct was lower than those of the other mutants, it was still
significantly higher than the control (130 versus 20 pmol/mg/min; Fig. 3). Thus, the Ca2+ sensitivity, from both
the in vivo and in vitro assays, of all of the
mutants agree well and confirm that the C2b region is critical for the Ca2+ sensitivity of ACVIII. However, it is notable
that the activities of C2
1184-1248 and
NC2
1-106,
1184-1248 are lower than that of
the wild type in vitro (Fig. 3), while in the intact cells
in the presence of forskolin, their activity is much higher than the
wild type activity (Fig. 2B). This discrepancy could result
from (among other things) misdirection of the adenylyl cyclase
constructs to inappropriate membrane locations. Therefore, to confirm
the expression of all of the mutants and to estimate the amount of
protein being expressed in the plasma membrane, Western blot
experiments were performed on the membranes that were used in in
vitro assays.

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Fig. 3.
In vitro measurement of the
Ca2+ sensitivity of various mutants. Adenylyl cyclase
activity was determined in plasma membranes (5 µg/reaction) prepared
from cells transfected with either vector alone (pcDNA3.0, as
control), wild type ACVIII (AC8), or the mutants indicated as described
under "Experimental Procedures." Activity was measured in the
presence of calmodulin (1 µM) and forskolin (20 µM), with (white bars) or without
(black bars) 2 µM free
Ca2+. Mutant numbers from Table I are used. Values shown
are from an experiment that was repeated at least three times with
similar results.
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Expression of Mutants Assessed by Western Blot--
Ab VIII-A
1229-1248 was used to recognize the mutants with intact C termini
(Fig. 4A), while Ab VIII-A
666-682 was used to recognize the mutants lacking C termini but with
intact C1b regions (Fig. 4B). Mutants 1-13 were all
expressed, although in varying amounts (Fig. 4, A and
B). Mutants N
1-106, N
1-49, N
1-106,
158-169,
C1
620-644, and NC1
1-106,
620-644
were expressed well (Fig. 4A), which coincided with their
approximately equivalent in vitro activity. On the other
hand, C2
1126-1248 was well expressed as
determined by Western blot but only had background level activity,
indicating that this molecule is totally inactive (Fig. 4B).
C1
635-700 and
NC1
1-106,
635-700 were also well expressed
(Fig. 4A), but their in vitro activity was
low, underlining their low intrinsic activity. Deletion mutants C1
588-619, NC1
1-106,
588-619, C2
1184-1248, and
NC2
1-106,
1184-1248 were shown to have low
expression in the plasma membrane (Fig. 4, A and
B), which corresponded to their low activity in
vitro. Based on these Western blot results, it is conceivable that
the discrepancy between the activities of
C2
1184-1248 and NC2
1-106,
1184-1248, which were high in
vivo but low in vitro, could reflect mistargeting of
these two mutated proteins to subcellular fractions not banding as the plasma membrane in our preparation.

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Fig. 4.
Western blots of various constructs. The
same membrane proteins assayed in Fig. 3 were loaded on
SDS-polyacrylamide gel (10 µg/lane) and probed as described under
"Experimental Procedures." Mutant numbers (Table I) are indicated
at the top of the corresponding lanes. The
molecular masses are marked in kDa on the left.
A, Ab VIII 1229-1248 antibody was used to probe the mutants
with intact C termini. B, Ab VIII-A 666-682 was used to
probe the mutants with intact C1b regions but with a deleted C
terminus. This result is representative of two experiments yielding
similar results.
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Characterization of the Three Deletions,
N
1-106, C2
1184-1248, and
NC2
1-106,
1184-1248--
From the foregoing data,
three of the apparently more informative mutants,
N
1-106, C2
1184-1248, and NC2
1-106,
1184-1248, were chosen for more detailed
analysis. Ca2+ and calmodulin concentration-response curves
were generated for these three mutants and compared with the wild type
ACVIII. For wild type ACVIII, activity began to increase at 0.4 µM Ca2+, reached a plateau between 1 and 10 µM, and declined when the free Ca2+
concentration exceeded 10 µM (Fig.
5A). The decrease of ACVIII activity at high [Ca2+]free is considered to
reflect Ca2+ competing with Mg2+ at an
allosteric regulatory site (1, 40, 41). This inhibition by high
[Ca2+] is a property of all mammalian adenylyl cyclases,
apart from AC3, regardless of their response to Ca2+ in the
submicromolar range (25, 41, 42). The Ca2+
concentration-response curve of N
1-106 is similar to
that of the wild type, except that the Ca2+-stimulated
activity is somewhat higher (Fig. 5A). On the other hand,
the C2
1184-1248 construct was unaffected by low [Ca2+]free, although its activity started to
decline when [Ca2+]free exceeded 10 µM (Fig. 5A). Ca2+ also did not
stimulate, but rather inhibited, the activity of NC2
1-106,
1184-1248 when concentrations surpassed
1 µM (Fig. 5A).

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Fig. 5.
Ca2+ and
calmodulin concentration-response curves of three deletion
mutants, N 1-106,
C2 1184-1248, and
NC2 1-106, 1184-1248,
and wild type ACVIII. Adenylyl cyclase activities were
determined in plasma membranes prepared from cells transfected with
wild type ACVIII ( ), N 1-106 ( ),
C2 1184-1248 ( ), or
NC2 1-106, 1184-1248 ( ). A,
Ca2+ concentration-response curves in the presence of 2 µM forskolin, 1 µM calmodulin, and the
indicated free Ca2+ concentrations. -Fold stimulation of
adenylyl cyclase is indicated for each Ca2+ concentration
relative to activity measured in the absence of Ca2+.
B, as A except that exogenous calmodulin was not
included in the reactions. C, calmodulin
concentration-response curves in the presence of 2 µM
forskolin, 22.4 µM free Ca2+ and the
indicated exogenous calmodulin concentrations. -Fold stimulation of
adenylyl cyclase is indicated for each calmodulin concentration
relative to activity measured in the absence of calmodulin.
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Ca2+ concentration-response experiments were also performed
in the absence of calmodulin. The wild type ACVIII was stimulated by
approximately 50% with supramicromolar Ca2+ (Fig.
5B). This stimulation apparently resulted from residual calmodulin in the plasma membrane, although the preparation was washed
twice with assay buffer containing 800 µM EGTA. Such
persistence of calmodulin with adenylyl cyclase has been long
encountered (41). It had been considered to reflect the persistent
association of calmodulin with plasma membranes or the presence of
calmodulin in other assay components, e.g. albumin or
creatine phosphokinase (43). However, it is conceivable that there is a
tight binding site for calmodulin on adenylyl cyclases. The latter
possibility is supported by two observations: (i) the Ca2+
stimulation of wild type ACVIII observed without the addition of
exogenous calmodulin can be abolished by the peptide 8Ncam, which binds
to calmodulin (data not shown), and (ii) without exogenous calmodulin, Ca2+ has little effect on
N
1-106 (Fig. 5B). Thus, the deletion of
amino acids 1-106 appears to result in no residual calmodulin. As
expected, no stimulation was seen with either
C2
1184-1248 or
NC2
1-106,
1184-1248, and activities started to
decline at 1 µM free Ca2+ concentration (Fig.
5B).
Calmodulin concentration-response experiments were performed
on the mutants in the presence of 22.4 µM free
Ca2+. (A high [Ca2+] was chosen to saturate
high concentrations of calmodulin.) N
1-106 elicited a
similar stimulatory profile as wild type ACVIII, although the -fold
stimulation (approximately 6-fold) was higher than that of the wild
type (approximately 4-fold; Fig. 5C). Deletions
C2
1184-1248 and
NC2
1-106,
1184-1248 were relatively insensitive to
calmodulin when the concentration was lower than 1 µM
(Fig. 5C). However, when the calmodulin concentration was increased to 10 µM, the activities of these two mutants
were about 2-fold higher than the activity in the absence of
calmodulin (Fig. 5C). This increase in the activities of
C2
1184-1248 and
NC2
1-106,
1184-1248, in the presence of a
high concentration of calmodulin, most likely reflected a reversal of
the inhibitory effects of high (22.4 µM) Ca2+
on these mutants (see Fig. 5B) due to calmodulin chelation
of Ca2+.
Peptide Competition Studies--
The calmodulin overlay assay data
and the mutagenesis screen identified two putative
calmodulin-binding sites on ACVIII; one is in the N terminus,
amino acids Met1 to Ser110, and one is in the C
terminus, amino acids Pro1184 to Pro1248. Using
the Chou and Fasman secondary structure prediction program, we located
only one putative site in the N terminus that could form a helical
structure - from Gln34 to His52. This site
resembles a classic Ca2+-dependent
calmodulin-binding site (Fig.
6A; see Ref. 18), in that it
has basic amino acids (net charge, +4) and two aromatic amino acids,
Trp38 and Phe50. The hydrophobic amino acid
distribution pattern is also similar to some known
Ca2+-dependent calmodulin-binding sites, such
as the death-associated protein kinase (a serine/threonine kinase
associated with mediation of interferon-induced cell death (19)), the
human erythrocyte Ca2+ pump (20), a
Ca2+-regulated nitric-oxide synthase (16), rabbit skeletal
muscle myosin light chain kinase (21), the inositol 1,4,5-trisphosphate type I receptor, and Bacillus anthracis adenylyl cyclase
(Fig. 6A; Ref. 18). At the C terminus of ACVIII, between
amino acids Pro1184 and Pro1248, the best
candidate for a calmodulin-binding site would be from amino acid
Tyr1187 to Glu1211. This site is similar to the
IQ motif (18), although it lacks one glycine residue (Fig.
6B). It also tends to form a helical structure as predicted
by the Chou and Fasman analysis and shares homology with other
calmodulin-binding sites, such as the ones in inositol
1,4,5-trisphosphate receptor (18), the
-subunit of protein kinase C
(18), and the
-subunit of cyclic nucleotide-gated channel (Ref. 23;
Fig. 6B). This site would be an atypical calmodulin-binding site if it indeed bound calmodulin, since the IQ motif normally binds
calmodulin independently of Ca2+, although some studies
have shown that the IQ motif can also bind to calmodulin in a
Ca2+-dependent manner (22, 23). Two peptides
from ACVIII were synthesized for competition studies, one corresponding
to amino acids Gln34 to His52, termed 8Ncam,
the other corresponding to amino acids
Tyr1187-Glu1211, termed 8Ccam. The N termini
of the two peptides were protected by acetylation, and the C termini
were protected by amidation to prevent self-circulation of the peptide
and to mimic the in vivo conformation.

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Fig. 6.
Two calmodulin-binding sites located on
ACVIII by structure prediction and synthetic peptide studies.
A, prediction of a putative calmodulin-binding site in the
N-terminal fragment of ACVIII, which had bound calmodulin in overlay
assays (Fig. 1). Amino acid numbers are placed above the
sequence. The predicted secondary structure was obtained
using the Chou and Fasman program. The putative calmodulin-binding
site, called 8Ncam, is indicated in boldface
type. The sequence of 8Ncam is aligned with those of
calmodulin-binding sites from death-associated protein (DAP)
kinase (a serine/threonine kinase associated with mediation of
interferon-induced cell death), the human erythrocyte Ca2+
pump, Ca2+-regulated nitric-oxide synthase (NO
synthase), rabbit skeletal muscle myosin light chain kinase
(MLCK), and B. anthracis adenylyl cyclase. Their
hydrophobic amino acid patterns and net charges are compared.
B, prediction of a putative calmodulin-binding site in the C
terminus of ACVIII whose binding to calmodulin was suggested by
mutagenesis studies. The secondary structure of amino acids 1182-1248
of ACVIII was predicted using the Chou and Fasman program. The putative
calmodulin-binding site is indicated with boldface
type. The sequences of IQ motif, 8Ccam, IP3 receptor,
protein kinase C ( -subunit), and CNG channel ( -subunit) are
aligned, and important amino acids are compared. C, the
effects of peptides on the calmodulin responsiveness of ACVIII. Plasma
membranes from HEK 293 cells transfected with wild type ACVIII were
assayed in the presence of 2 µM forskolin, 7.74 µM free Ca2+, and the indicated calmodulin
concentrations, along with vehicle (water, ), 8Ncam (4 µM, ), CamkII (4 µM, ), 8Ccam (4 µM, ), or 8CT (4 µM, ). (ACVIII
activities in the presence of different peptides, in the absence of
Ca2+ and exogenous calmodulin, are also shown.)
D, inhibition profiles of various peptides. Plasma membranes
from HEK 293 cells transfected with ACVIII. Activities were assayed in
the presence of 2 µM forskolin, 7.74 µM
free Ca2+, 0.3 µM calmodulin, and the
peptides at the indicated concentrations (8Ncam, ; CamkII, ;
8Ccam, ; and 8CT, ). IC50 values, extracted from the
fitted inhibition curves as described under "Experimental
Procedures," were 1.15 ± 0.25 µM for 8Ncam,
0.65 ± 0.17 µM for 8Ccam, and 0.15 ± 0.1 µM for CamkII.
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These peptides were used in in vitro assays in
competition studies. A calmodulin concentration-response curve was
generated for ACVIII, in the presence or absence of added peptides (at
4 µM). CamkII, the peptide corresponding to the
calmodulin-binding site of CAM kinase II, was used as a positive
control. 8CT, the peptide corresponding to the C terminus of ACVIII,
from Thr1229 to Pro1248, was used as a negative
control. In the presence of 1 µM calmodulin and 7.74 µM free Ca2+, 8CT had no effect on the
calmodulin concentration-response curve, while CamkII suppressed the
ACVIII activity and shifted the curve to the right (Fig.
6C). Both the 8Ncam and 8Ccam peptides also suppressed the
calmodulin stimulated activity of ACVIII, although not quite as
efficaciously as the CamkII peptide (Fig. 6C).
Peptide inhibition experiments were also performed for these four
peptides in the presence of a fixed calmodulin concentration (0.3 µM; and 7.74 µM free Ca2+).
Again, 8CT had no effect on ACVIII activity even at its highest concentration (Fig. 6D). CamkII began to inhibit ACVIII
activity when its concentration exceeded 0.1 µM (Fig.
6D; IC50, 0.15 ± 0.1 µM;
n = 3). 8Ncam began to inhibit ACVIII activity at 0.3 µM, (IC50, 1.15 ± 0.25 µM; n = 3; Fig. 6D). 8Ccam was
a little more potent than 8Ncam (IC50, 0.65 ± 0.17 µM; n = 3; Fig. 6D). These
experiments clearly demonstrate that both peptide sequences derived
from the N and C termini of ACVIII are bona fide calmodulin-binding sequences and that the most potent peptide is also the one that is of
most functional significance.
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DISCUSSION |
This study has explored the calmodulin-binding sites on
ACVIII. The related Ca2+-stimulable ACI binds calmodulin in
the C1b region of the molecule. However, ACVIII and ACI are very
dissimilar (only 40% homologous) in this region. Therefore, it might
not have been unexpected that different sites would mediate the
Ca2+ stimulation of ACVIII. Calmodulin overlay assays
revealed one putative Ca2+-dependent
calmodulin-binding site in the N terminus of ACVIII (Fig. 1). However,
without this region the enzyme was still sensitive to Ca2+
(Figs. 2 and 3). On the other hand, using mutagenesis and functional assays, only those mutants lacking the C2b region, such as
C2
1184-1248 and NC2
1-106,
1184-1248, could not be stimulated by Ca2+
either in vivo or in vitro (Figs. 2 and 3). This
suggests that the C-terminal region is responsible for the
Ca2+/calmodulin stimulation of ACVIII. Moreover, the high
basal activities of C2
1184-1248 and
NC2
1-106,
1184-1248 suggests the removal of
autoinhibitory domains (the C2b region playing the major role), which
suppress the activity of wild type ACVIII. The binding of
Ca2+/calmodulin to the autoinhibitory domain apparently
relieves the inhibitory binding and activates the enzyme. Such a
disinhibitory mechanism is employed in a number of
Ca2+/calmodulin-activated enzymes, such as
Ca2+-regulated nitric-oxide synthase (44),
Ca2+/calmodulin-dependent protein kinases
(45-47), a Na+/H+ exchanger (48), and
calcineurin (49). It appears as though most of the C2b region might
participate as the inhibitory binding domain, since the two synthetic
peptides 8Ccam (25 residues) and 8CT (20 residues) from the C2b region
did not inhibit ACVIII activity in the absence of Ca2+ and
calmodulin (Fig. 6C). This disinhibition mechanism of
Ca2+/calmodulin stimulation is different from that of
forskolin, which is thought to stabilize the C1/C2 heterodimer to
activate the adenylyl cyclase activity (9, 10). The latter suggestion is supported by the fact that forskolin and Ca2+/calmodulin
synergistically stimulate ACVIII.
The putative calmodulin-binding site in the C terminus has the
signature sequence of an IQ motif, which generally reflects Ca2+-independent calmodulin binding. It is possible that
the binding of calmodulin is Ca2+-independent and that
Ca2+ binding changes the conformation of bound calmodulin
to activate the enzyme. Alternatively, the binding of calmodulin may be
Ca2-dependent as is the case with the
-subunit of rod photoreceptor cyclic nucleotide-gated channel (23).
It is of some interest that the peptide synthesized from this region
(8Ccam) is only 5 times less effective than 8CamkII, which is a
conventional calmodulin-binding peptide. This observation underscores
how much we still need to learn about the molecular characteristics of
calmodulin-binding sequences.
The putative calmodulin-binding site for the N terminus of ACVIII is a
conventional Ca2+-dependent calmodulin-binding
site, which is reinforced by the results of overlay assays. The fact
that the double deletion NC2
1-106,
1184-1248 has
higher activity in vivo (Fig. 2) and can be inhibited by a lower concentration of Ca2+ (Fig. 5A) than
C2
1184-1248 suggests that this site contributes to the
Ca2+ stimulation of ACVIII, although this contribution must
be minor.
The fact that there is apparently more residual calmodulin in ACVIII
wild type membrane preparations than in those of N
1-106 (Fig. 5B) might suggest a role of the N terminus of ACVIII
as a Ca2+-independent calmodulin trap, notwithstanding the
apparently conflicting evidence of the
Ca2+-dependent manner of the N-terminal
calmodulin-binding site from overlay assays.
Unlike the two regulatory domains (the N terminus and the C2b
region) discussed above, the C1b region does not have a free end, which
suggests that the disruptions on this region could more easily change
the activity of adenylyl cyclases. However, continuous deletions in the
C1b region of ACVIII could not eliminate the Ca2+
stimulation of ACVIII (Fig. 2A), while, by contrast, a point mutation in this region of ACI abolished its Ca2+
sensitivity (17). The different calmodulin-binding sites on ACVIII and
ACI are underlined by some differences in their regulation by
Ca2+/calmodulin; for instance, ACI is more sensitive to
lower concentrations of Ca2+ than is ACVIII, and ACVIII is
more stimulable by Ca2+/calmodulin than ACI (25). The
calmodulin-binding site in ACI is rich in basic amino acids (net charge
is +7), and the binding is Ca2+-dependent (15,
16). Since no hyperactivity was observed by mutating the C1b region on
ACI (15, 17) and the movement of the C1b region is likely to be more
restrained than those of the N terminus and C terminus, the mechanism
of Ca2+/calmodulin regulation of ACI might be to stabilize
the C1/C2 heterodimer, as has been proposed for forskolin and
Gs
a (9, 10), unlike the disinhibitory mechanism we have
proposed for ACVIII.
In conclusion, two calmodulin-binding sites exist on ACVIII, one (at
the C terminus) is of profound regulatory significance, whereas the
other (at the N terminus) plays a more minor role. Whether these two
domains of ACVIII physically interact to share the same molecule of
calmodulin remains to be determined in future studies. Given that the
C1a and C2a regions clearly interact for catalytic activity (4, 5, 7,
9, 10, 35), the tantalizing possibility that adenylyl cyclase could
adopt a transporter-like structure (11, 50) would be greatly
strengthened by interactions between the N and C termini.