From the Department of Neurobiology, Pharmacology,
and Physiology, The University of Chicago, Chicago, Illinois 60637 and
the § Department of Pharmacology, Mount Sinai School of
Medicine, New York, New York 10029
Received for publication, November 15, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Mammalian membrane-bound adenylyl cyclase
consists of two highly conserved cytoplasmic domains (C1a and C2a)
separated by a less conserved connecting region, C1b, and one of two
transmembrane domains, M2. The C1a and C2a domains form a catalytic
core that can be stimulated by forskolin and the stimulatory G protein
subunit Changes in intracellular cAMP concentration regulate a variety of
physiological responses, such as sugar and lipid metabolism, olfaction,
and cell growth and differentiation. Cyclic AMP directly activates
diverse molecules including cAMP-dependent protein kinase, cyclic nucleotide-gated Na+/Ca2+ and
Na+/K+ channels, and cAMP-regulated guanine
nucleotide exchangers of Rap-1 (1-3). Intracellular cAMP concentration
is controlled primarily through regulation of adenylyl cyclase. To
date, there are nine isoforms of membrane-bound adenylyl cyclase and
one soluble adenylyl cyclase found in mammals (4-6). Each isoform of
adenylyl cyclase has its own pattern of tissue distribution and unique
regulation to fit its physiological roles.
All nine isoforms of mammalian membrane-bound adenylyl cyclases share a
common structure including two ~40-kDa cytoplasmic domains (C1 and
C2), each following an ~20-kDa hydrophobic domain (M1 and M2) (see
Fig. 1) (4, 5). When expressed separately and then mixed in
vitro, the conserved portions of the C1 and C2 domains, C1a and
C2a, form a soluble enzyme that exhibits most of the regulatory
properties of the membrane-bound enzyme (7-22). This soluble enzyme
can be activated by subunit Such soluble enzyme models have yielded high resolution structures of
the C2a homodimer of AC2 (2C2a) and the 5C1a/2C2a heterodimer (23, 24).
These structures suggest a catalytic mechanism and basic mode of
regulation. The catalytic site of adenylyl cyclase is formed in one of
two small pockets at the interface of C1a and C2a. Forskolin binds the
other pocket to shift the alignment of C1a and C2a. The activation of
extracellular signals, such as hormones and neurotransmitters, triggers
the exchange of guanine nucleotide in G The C1b region links C1a to the M2 region of mammalian adenylyl cyclase
and is far less conserved than the C1a and C2a regions among the nine
isoforms of membrane-bound adenylyl cyclase (Fig. 1). Although C1b is not required for
catalysis, it is essential in several regulatory functions, such as
calmodulin activation of AC1 and cAMP-dependent protein
kinase modulation of AC6 (27-30). In this paper, we use the
soluble adenylyl cyclase from the C1a and C2a domains of AC7 and its
membrane-bound counterpart to address the regulatory role of AC7
C1b.
Materials--
Forskolin was from Calbiochem.
Restriction enzymes and Vent DNA polymerase were from New England
Biolabs (Beverly, MA). The Bradford reagent was from Bio-Rad.
Renaissance Western blot Chemiluminescence Reagent Plus and
[ Plasmids--
The coding sequence for AC7 C1b was amplified by
18 cycles of polymerase chain reaction using the human AC7 as
the template, Vent DNA polymerase, and primers
5'-GGTCGAATTCCCCGGAGCCAGCAGCCACCCCCGCCCAGCC-3' and
5'-CGCAAAGCTTCTAGTGGCGGGCCCGGGGGATGGGTGCCAG-3' (7C1b from amino
acids 483 to 596). The resulting DNAs were digested with EcoRI and HindIII and ligated into pProEx-HAH6,
which was digested with the same enzymes, resulting in
pProEx-HAH6-7C1b. Plasmid used to express 7C1b(E582A) was constructed
by Quick-change site-directed mutagenesis using pProEx-HAH6-7C1b as the
template. Mutagenic oligonucleotides contained 25-30 complementary
nucleotides flanking each side of the codon(s) targeted for mutation.
Mutations were confirmed by DNA sequencing.
The yeast two-hybrid vectors, pLexA-W1 and pB42AD-W1, were constructed
by Quick-Change site-directed mutagenesis using pLexA and pB42AD as the
templates (CLONTECH) so that the coding of the DNA
binding domain of LexA and activating domain of B42 can fuse in-frame
with the multiple cloning site of the pProEx-HAH6 vector. To construct
pLexA-W1-7C1a and pLexA-W1-7C2a, the HindIII-blunted EcoRI fragments of 7C1a and 7C2a coding sequences were
excised from pProEx-HAH6-7C1a and pProEx-HAH6-7C2a and ligated into
pLexA-W1, which was digested with EcoRI and
PvuII. A similar strategy was used for constructing
pB42AD-W1-7C1b except that the filled in XhoI site of
pB42AD-W1 was used.
Expression and Purification of 7C1b-SlyD Protein--
To express
7C1b and 7C1b mutants, pProEx-HAH6-7C1b and pProEx-HAH6-7C1b(E582A)
were transformed into Escherichia coli strain BL21(DE3), and
the cells were grown in T7 medium containing 50 µg/ml ampicillin at
30 °C. When A600 reached 0.4, isopropyl-1-thio- Purification of 7C1b Protein from E. coli slyD
Purification of SlyD and SlyD Mutants--
To express SlyD and
its mutant proteins, the plasmids for the expression of wild type SlyD
and SlyD mutants (L11R and Adenylyl Cyclase Assay--
Adenylyl cyclase assays were
performed in the presence of 10 mM MgCl2 and
0.5 mM ATP at 30 °C for 20 min as described previously (7). To express wild type and mutated forms of G Peptide Synthesis--
Peptides were synthesized with an Applied
Biosystems model 430A peptide synthesizer. Identity of the peptides was
verified by mass spectrometry. Peptides were dissolved in water and
used at final concentration as indicated.
Characterization of 7C1a and 7C2a--
We made C1a (amino acids
263-476) and C2a (amino acids 864-1080) from human AC7 that
effectively could be expressed and purified (Fig.
2A) (34). To compare soluble
adenylyl cyclase with its native enzyme, a recombinant baculovirus was
constructed to express membrane-bound AC7 (34). Both membrane-bound AC7
and soluble 7C1a-7C2a complex were substantially stimulated by
G Purification and Characterization of 7C1b-SlyD--
The soluble
7C1a-7C2a complex is used to address the role of C1b because it does
not contain C1b. We constructed a vector to express a polypeptide chain
from amino acids 457 to 596 of human AC7 named 7C1b. Adding lysates of
E. coli containing 7C1b, we observed 30% inhibition in
G
C1b contains a short stretch of sequence that is relatively conserved
among nine isoforms of adenylyl cyclase (Fig. 1). We made 7C1b mutants
with single point mutations in this region and screened for one that
expressed similar to wild type 7C1b but lost its ability to inhibit
7C1a-7C2a activity. Upon assaying E. coli lysates
containing 7C1b mutants, we found that 7C1b(E582A), a mutant with a
conserved glutamic acid at residue 582 that changed to alanine,
fit these criteria (Fig. 1, data not shown). We characterized both wild
type 7C1b and 7C1b(E582A) in our subsequent study.
7C1b and 7C1b(E582A) proteins were purified through three
chromatographic steps. After purification through Ni-NTA, Q-Sepharose, and Superdex 200 columns, the expected 16-kDa 7C1b and 7C1b(E584A) were
obtained with the yield of 2.2 mg from 1 liter of E. coli culture (Fig. 2B). A 25-kDa protein was copurified with 7C1b
at about 4-fold molar excess after purification through the Ni-NTA column. A stoichiometric quantity of 25 kDa and 7C1b was obtained after
purification through the Q-Sepharose and Superdex 200 gel permeation
columns. Based on the gel permeation column, the molecular size of 7C1b
is approximately 40 kDa, which is not a high molecular mass
aggregate. This suggests that 7C1b forms a complex with the 25-kDa protein in a 1:1 stoichiometric ratio. The N-terminal protein sequence analysis of the 25-kDa protein resulted in
XXVAXDLLVVLAYQ that matched the N-terminal
sequence of SlyD. SlyD, a 25-kDa protein with a histidine-rich
C-terminal region, can bind the Ni-NTA column effectively (38, 39). The
predicted pI of SlyD and 7C1b are 4.8 and 8.5, respectively, which may
lead to the charge-charge interaction of these two proteins. In the
subsequent study, we refer to our purified 7C1b and 7C1b(E582A)
preparations as 7C1b-SlyD or 7C1b(E582A)- SlyD.
SlyD Inhibits the Activity of Type 7 Adenylyl Cyclase--
SlyD
protein, one of several gene products originally identified as
suppressor of phage lysis, is a member of the
FK506-binding protein family and exhibits
cis-trans peptidylprolyl isomerase (PPIase)
activity (38, 39). We tested whether SlyD protein could affect the
activity of the 7C1a-7C2a complex using purified SlyD protein (Figs.
2C and 4). In the presence of
G
We then tested whether SlyD could modulate the activity of
membrane-bound adenylyl cyclases. Using the membrane preparations from
Sf9 cells that expressed individual isoforms of adenylyl cyclase, we observed that SlyD protein could inhibit the activity of
both AC2 and AC7 while having little effect on the activity of AC1.
Interestingly, SlyD could increase the activity of AC5 and AC6 (Fig.
5A). These effects could not
be observed with the PPIase-defective mutant, SlyD(L11R) (Fig.
5B). Thus, we conclude that the effect mediated by SlyD is
both isoform-specific and involves PPIase activity.
Inhibition in the Activity of Soluble 7C1a-7C2a by 7C1b-SlyD and
7C1b--
To address the role of 7C1b in regulating AC7, we used
7C1b-SlyD and 7C1b(E582A)-SlyD. As a control, we used an equal quantity of purified SlyD compared with that in our 7C1b-SlyD preparations and
based on protein concentration and SDS-PAGE. In the concentration range
of 0.1-0.6 µM G
To demonstrate 7C1b inhibition in the absence of SlyD, we expressed and
purified 7C1b in RY3041, an E. coli BL21(DE3) strain that is
defective in the expression of SlyD. Expression of 7C1b in the
slyD Inhibition of Membrane-bound Adenylyl Cyclases by Peptides Derived
from C1b--
To address the role of 7C1b further, we made 28-mer
peptides derived from the most conserved region of C1b across three
adenylyl cyclases (Fig. 1). FGSI and ISLL peptides (named by their
first four amino acid sequences) were derived from two closely related adenylyl cyclases AC7 and AC2, respectively. FGSI peptide inhibited the
adenylyl cyclase activity of 7C1a-7C2a activated by forskolin and/or
G Interaction of 7C1b with 7C1a and 7C2a--
To initially assess
interaction of 7C1b to 7C1a and/or 7C2a, we used the yeast two-hybrid
system where transactivation of the lacZ reporter indicates potential
interaction (40). We observed light blue colonies on medium containing
5-bromo-4-chloro-3-indolyl The catalytic core of mammalian adenylyl cyclase consists of two
cytoplasmic domains, C1a and C2a (22). The catalytic site is located at
the interface of C1a and C2a and includes residues from both domains
(24, 26). The activation of adenylyl cyclase does not seem to involve a
major conformational change in either C1a or C2a upon the binding of
G The C1b region joins C1a to C2a by connecting C1a to the second
transmembrane domain (M2), which then connects to C2a (Fig. 1). Thus,
C1b is in an ideal location to dynamically regulate the interaction
between C1a and C2a and thus modulate the catalytic rate. Using both
C1b protein and peptides derived from C1b, our data show that AC7 C1b
can inhibit AC7. We also observed that both C1b and peptides derived
from C1b inhibit soluble 7C1a-7C2a more potently than they inhibit
membrane-bound AC7. Such a difference is probably due to the presence
of endogenous C1b in the membrane-bound AC7.
How does C1b inhibit the activity of AC7? Our yeast two-hybrid analysis
suggests that AC7 C1b can bind 7C1a and 7C2a. Using 7C1b-SlyD, our
coimmunoprecipitation experiment shows that 7C1b can bind both 7C1a and
7C2a, which is in agreement with our yeast two-hybrid
analysis.2 Using antibody
that can immunoprecipitate 7C2a, the active 7C1a-7C2a-G C1b is one of the key regions for isoform-specific regulation. An
amphipathic region that is only present in AC1 C1b is involved in the
binding and activation of Ca2+/calmodulin (27, 28, 30).
Splicing variants of AC8 that have different C1b sequences are
differentially sensitive to calmodulin (41). AC6 C1b has a
cAMP-dependent protein kinase phosphorylation site that
confers feedback inhibition by cAMP (29). The C1b region may also be
involved in inhibition by calcium and G We were surprised to find that the catalytic activity of certain
isoforms of mammalian adenylyl cyclase can be stimulated or inhibited
by SlyD. Using mutant forms of SlyD, we find that this isoform-specific
regulation may be due to PPIase activity of SlyD. This raises the
possibility that mammalian PPIases may regulate the activity of
selective isoforms of mammalian adenylyl cyclase directly. There are
three gene families of eukaryotic PPIases: FK506-binding protein,
cyclophilins, and parvulins (44). PPIase is known as a folding helper
enzyme; however, growing evidence suggests that PPIases may serve to
regulate biological activity after the proper folding of proteins (31,
45, 46). Thus, an understanding of how SlyD regulates mammalian
adenylyl cyclases may not only provide a novel molecular tool for
modulating adenylyl cyclase activity in an isoform-specific manner but
may also lead to a new area of research in the regulation of adenylyl
cyclase activity by PPIases.
(G
s). In this study, we analyzed the
regulation of type 7 adenylyl cyclase (AC7) by C1b. The C1a, C1b, and
C2a domains of AC7 were purified separately. Escherichia
coli SlyD protein, a cis-trans peptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b). SlyD
protein can inhibit the G
s- and/or forskolin-activated
activity of both soluble and membrane-bound AC7. Mutant forms of SlyD
with reduced PPIase activity are less potent in the inhibition of AC7 activity. Interestingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein,
raising the possibility that mammalian PPIase may regulate enzymatic
activity of mammalian adenylyl cyclase. Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than
SlyD alone. This inhibition by 7C1b is abolished in a 7C1b mutant in which a conserved glutamic acid (amino acid residue 582) is
changed to alanine. Inhibition of adenylyl cyclase activity by 7C1b is further confirmed by using 7C1b purified from an E. coli
slyD-deficient strain. This inhibitory activity of AC7 is also
observed with the 28-mer peptides derived from a region of C1b
conserved in AC7 and AC2 but is not observed with a peptide derived
from the corresponding region of AC6. This inhibitory activity
exhibited by the C1b domain may result from the interaction of 7C1b
with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
of G protein (G
s)1 and by
the diterpene, forskolin, and can be inhibited by subunit
of
Gi and by adenosine analogs termed P-site inhibitors.
s, resulting in
conformational changes in the switch regions of G
s. The
switch 2 region of G
s interacts with both C2a (
2 and
3/
4 regions) and C1a (N-terminal loop of
1) to promote the
proper alignment between C1a and C2a (9, 24). Amino acid residues from
both C1a and C2a domains contribute to the "two-metal"- mediated
catalysis, a catalytic mechanism also used in many DNA polymerases and
nucleotidyltransferases (25, 26). Proper alignment of C1a and C2a
induced by forskolin and G
s facilitates catalysis.
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Fig. 1.
Model of mammalian adenylyl cyclases
(left) and sequence comparison of the conserved
portion of C1b regions among nine isoforms of mammalian adenylyl
cyclase (right). The sequences of three peptides,
ISLL, FGSI, and FLLT, derived from the C1b region of rat AC2, human
AC7, and rat AC6, respectively, are compared with the C1b of six other
isoforms of adenylyl cyclase. The conserved sequences are
highlighted in bold. The asterisk
symbol marks the highly conserved glutamic acid at residue
582 of human AC7.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP were from PerkinElmer Life Sciences. Ni-NTA
resin was from Qiagen (Chatsworth, CA). Quick-Change kit for
site-directed mutagenesis was from Stratagene (La Jolla, CA). Big-dye
kit for automatic DNA sequencing was from PerkinElmer Life Sciences.
Reagents for yeast two-hybrid analysis were from
CLONTECH (Palo Alto, CA).
-D-galactopyranoside was added to 30 µM final concentration. After 4 h, the induced cells
were collected and lysed. The recombinant proteins were purified using
Ni-NTA and Q-Sepharose columns similar to the purification of 1C1a and
2C2a as described (7). E. coli SlyD was copurified with
7C1b. 7C1b-SlyD and 7C1b(E582A)-SlyD complexes were further purified
using Amersham Pharmacia Biotech Superdex 200 column and identified
based on molecular weight and immunoblot. The concentration of proteins
was determined using the Bradford reagent with bovine serum albumin as
standard (32).
Strain--
To obtain 7C1b free of SlyD,
pProEx-HAH6-7C1b was transformed into E. coli RY-3041, a
BL21(DE3) slyD
strain, and grown to
A600 = 0.45. For induction,
isopropyl-1-thio-
-D-galactopyranoside was added to 30 µM, and the incubation temperature was lowered to
22 °C. Cells were harvested 4 h after induction. Cells were lysed in buffer containing 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 0.025% Triton X-100, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1 µM pepstatin A, 0.5 µg/ml aprotinin, 0.1 mM benzamidine, and 0.1 mg/ml
lysozyme, followed by a 4-min sonication (1 s on, 3 s off) and
centrifugation at 100,000 × g for 30 min. Lysate was
loaded on a Ni-NTA affinity column (10 ml of resin) pre-equilibrated to
the same buffer as used for lysis (without lysozyme). The column was
washed with 100 ml of low imidazole (same buffer with 15 mM imidazole) and then eluted with a 15-200 mM imidazole
gradient (3-min fractions and 2 ml/min flow rate). 7C1b was detected by Coomassie Blue staining of SDS-PAGE, and fractions containing relatively pure 7C1b were concentrated to 1 ml by Amicon positive pressure filtration with Millipore filter YM10 membrane. A combination of membrane dialysis (Spectra/Por 16 mm, molecular weight cut off 10,000) and Amicon filtration were used to bring NaCl and imidazole concentrations to less than 2 and 0.2 mM, respectively.
107-111) in plasmid pJF118EF were
transformed into E. coli BL21(DE3) wild type and
slyD
(MCX) strains, respectively. The resulting
cells were grown in modified T7 media with 50 µg/ml ampicillin at
30 °C to an A600 = 0.4. Protein expression
was then induced with 100 µM
isopropyl-1-thio-
-D-galactopyranoside. 19 h after
infection, 0.1 µM phenylmethylsulfonyl fluoride was added. The cells were then spun down, and the cell pellet was frozen at
80 °C. SlyD and its mutant proteins were purified by Ni-NTA and
Q-Sepharose column in the manner similar to that of 7C1b. The yields of
SlyD and its mutant proteins were about 10-20 mg with greater than
95% purity from each liter of E. coli culture.
s, pQE60
that carried wild type or mutant forms of G
s were
transformed to BL21(DE3) that carried pUBS520, and the induction and
purification of recombinant G
s were performed as
described previously (33). Recombinant G
s was activated
by 50 µM AlCl3 and 10 mM NaF.
Expression of soluble and membrane-bound AC7 and purification of 7C1a
and 7C2a were performed as described (34). Adenylyl cyclase activity of
Sf9 cell membranes was completed as described previously
(35).
-Galactosidase Assay--
Yeast EGY48 strain that carried
pSH18-34 was transformed with pLexA-W1-7C1a or pLexA-W1-7C2a. The
resulting transformants were then transformed with
pB42AD-W1-7C1b. The yeast clones that carried all three plasmids were
grown in synthetic defined media lacking uridine, histidine, and
tryptophan and containing 2% galactose and 1% raffinose at 30 °C
until the cells reached a density of A600 = 1.
-Galactosidase assay was performed with 1 ml of cells as described
previously (36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s and forskolin, consistent with the observation that a
coexpressed Gs-coupled receptor could raise cAMP formation
over 20-fold in mammalian cells overexpressing AC7 (34, 37).
Significant synergy was observed in both membrane-bound AC7 and soluble
7C1a-7C2a complex when G
s and forskolin were coapplied
(Fig. 3).
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Fig. 2.
Purified soluble AC7 domains and
SlyD. Purified 7C1a (HAH6-7C1a) and 7C2a
(H6-7C2) (A), 7C1b-SlyD complex (B),
and SlyD and its mutants (C) (2 µg each) were run onto
SDS-PAGE and stained by Coomassie Blue.
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Fig. 3.
Comparison of soluble 7C1a-7C2a and
membrane-bound AC7 activated by G s
and forskolin. Adenylyl cyclase activities of soluble 7C1a-7C2a
(A) and membrane-bound AC7 (mAC7) (B)
are shown. Adenylyl cyclase activity of 7C1a-7C2a (0.4 µM
each) and membrane-bound AC7 (80 µg) were activated by
G
s and forskolin. The concentration of G
s
in the assay is 240 nM. Sum (Fsk + G
s) is
the sum of adenylyl cyclase activity observed in the presence of
forskolin or G
s alone (
), Fsk + G
s is
adenylyl cyclase activity observed in the presence of both forskolin
and G
s (
). The means ± S.E. are representative
of three experiments. Fsk, forskolin.
s and forskolin-stimulated 7C1a-7C2a activity (data not
shown). However, no detectable 7C1b protein was found by immunoblot,
suggesting that 7C1b may undergo proteolysis at its N terminus. To test
this hypothesis, we constructed and expressed four N-terminal
truncation mutants of 7C1b. Lysates of E. coli containing
one of four mutants, 7C1b-
2 (amino acids 483-596 of human AC7),
exhibited 30% inhibition in 7C1a-7C2a activity stimulated by
G
s and forskolin and had the highest immunoreactivity. Thus, 7C1b-
2 was used in the subsequent study and renamed as 7C1b.
s, forskolin, or the combination of G
s
and forskolin, we observed up to 80% inhibition of adenylyl cyclase
activity by SlyD in a dose-dependent manner. To address
whether PPIase activity is required for this inhibition, we purified
and tested two SlyD mutant proteins, SlyD(L11R), which has no
detectable PPIase activity, and SlyD(
107-111), which has only 20%
PPIase activity (Fig. 2C) (47). SlyD(L11R) showed no
inhibition to 7C1a-7C2a, and SlyD(
107-111) had reduced potency, suggesting that PPIase activity plays a role in SlyD inhibition of
adenylyl cyclase (Fig. 4).
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Fig. 4.
Regulation of 7C1a-7C2a activity by
SlyD. 7C1a-7C2a (0.4 µM each) was stimulated by 2.2 µM G s (Gs
)
(A), 100 µM forskolin (Fsk)
(B), and 0.2 µM G
s with 100 µM forskolin (Gs
/Fsk)
(C) in the presence of the indicated SlyD concentration. The
specific activities of 7C1a-7C2a, stimulated by 2.2 µM
G
s, 100 µM forskolin, and 0.2 µM G
s with 100 µM forskolin,
were 130, 18, and 660 nmol·min
1·mg
1,
respectively. The means ± S.E. are representative of four
experiments.
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Fig. 5.
Regulation of membrane-bound adenylyl cyclase
by SlyD. The membranes of Sf9 cells containing the
indicated isoform of adenylyl cyclase (80 µg of each) were assayed
for their adenylyl cyclase activity in the presence of 100 µM forskolin, and 0.24 µM G s
was assayed in the presence of 20 µg of SlyD (A) and 20 µg of SlyD(L11R) (A and B). The data
shown represent adenylyl cyclase activities after subtracting out the
activity of membranes of Sf9 cells infected with control virus.
The specific activities of the control membranes in the absence and
presence of SlyD were 0.9 and 1.5 nmol·min
1·mg
1, respectively. The
means ± S.E. are representative of at least two
experiments.
s, 7C1b-SlyD exhibited
40-50% more inhibition than SlyD alone (Fig.
6A). The inhibition is
dose-dependent (Fig. 6B). Such inhibition in
excess of the SlyD control was not observed with 7C1b(E582A)-SlyD (Fig.
6). At a high concentration of G
s (2.2 µM)
or in the presence of both G
s and forskolin, no
C1b-mediated inhibition of 7C1a-7C2a was observed (Fig. 6A,
data not shown). When the activity of membrane-bound AC7 was analyzed,
we observed no additional effect of 7C1b-SlyD that was not accountable
by SlyD protein (data not shown).
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Fig. 6.
Adenylyl cyclase activity of 7C1a-7C1a
regulated by SlyD, 7C1b-SlyD, and 7C1b(E582A)-SlyD. A,
the effects of SlyD, 7C1b-SlyD, and 7C1b(E582A)-SlyD (5.3 µM each) on the activities of 7C1a-7C1a (0.4 µM each) in the indicated G s
concentration. Inset shows the effect of SlyD (solid
bar), 7C1b-SlyD (open bar), and 7C1b(E582A)-SlyD
(hatched bar) in the indicated G
s
concentration. Differences are statistically significant. *,
p = 0.002; +, p = 0.0005. B,
the effect on the activities of 7C1a-7C2a (0.4 µM each)
in the indicated concentrations of SlyD, 7C1b-SlyD, and
7C1b(E582A)-SlyD. Adenylyl cyclase assays were performed in the
presence of 0.6 µM G
s. The means ± S.E. are representative of six experiments.
strain was comparable with the expression
in the BL21(DE3) strain based on the immunoblot of the lysate.
Following the Ni-NTA column, we obtained 7C1b protein as seen on a
Coomassie Blue-stained SDS-PAGE gel and immunoblot. However, the
purified 7C1b could no longer be detected after only a few hours in the
cold room, making further purification steps impossible. After testing
a panel of stabilizing agents, we determined that 0.025% Triton X-100
in combination with 0.5 M NaCl stabilized the protein
sufficiently to conduct further concentration and buffer exchange
steps. We further determined that the salt concentration could be
reduced to < 2 mM if the protein was frozen in dry
ice quickly after salt removal and stored at
80 °C. This finding
allowed us to obtain 7C1b that is ~50% pure but free of SlyD (Fig.
7A). The partially
purified 7C1b inhibited the activity of 7C1a-7C2a (Fig.
7B). As a control for the detergent in the buffer, we boiled
our 7C1b preparation (100 °C for 3 min), which abolished the
inhibition (data not shown). This further supports an inhibitory role
for 7C1b.
View larger version (32K):
[in a new window]
Fig. 7.
Inhibitory effect of SlyD-free 7C1b on
adenylyl cyclase activity of soluble 7C1a-7C2. A,
purified 7C1b (1 µg) protein was run onto SDS-PAGE and stained by
Coomassie Blue. B, inhibition of adenylyl cyclase activity
of 7C1a-7C2a by purified 7C1b (1 µg). Adenylyl cyclase assay was
performed in the presence of 0.75 µM G s.
The means ± S.E. are representative of five experiments.
s (Fig. 8A).
Furthermore, the addition of FGSI and ISLL resulted in the inhibition
of adenylyl cyclase activity of soluble and membrane-bound AC7 in a
dose-dependent manner (Figs. 8B and 9A). FLLT peptide was derived
from the region corresponding to FGSI in a distantly related isoform,
AC6, and had little effect on the enzymatic activity of soluble and
membrane-bound AC7 (Figs. 8B and 9A). FLLT is
active because it inhibited the activity of membrane-bound AC6 as
described previously (Fig. 9B) (29). Interestingly, FGSI but
not ISLL was a potent inhibitor of membrane-bound AC6 (Fig.
9B). Taken together, these data indicate that C1b is capable of inhibiting the activity of adenylyl cyclase, and peptides from a
particular region of C1b may confirm isoform specificity.
View larger version (21K):
[in a new window]
Fig. 8.
Effects of ISLL, FGSI, and FLLT peptides
derived from the C1b region of AC7, AC2, and AC6 on adenylyl cyclase
activity of 7C1a-7C2a. The adenylyl cyclase activity of 7C1a-7C2a
(0.4 µM each) activated by 100 µM
forskolin, 2.2 µM G s, or 100 µM forskolin with 2.2 µM G
s,
in the presence of FGSI peptide (A) and 100 µM
forskolin with 0.24 µM G
s (B),
was performed in the indicated peptide concentration. The specific
activities of 7C1a-7C2a were 310 (2.2 µM
G
s), 850 (2.2 µM G
s and 100 µM forskolin), 22 (100 µM forskolin), and
660 (100 µM forskolin and 0.24 µM
G
s) nmol·min
1·mg
1. The
means ± S.E. are representative of at least three
experiments.
View larger version (19K):
[in a new window]
Fig. 9.
Effects of ISLL, FGSI, and FLLT peptides
derived from the C1b region of AC7, AC2, and AC6 on adenylyl cyclase
activity of membrane-bound AC7 (mAC7) and
membrane-bound AC6 (mAC6). The adenylyl cyclase
activity of mAC7 (A) and mAC6 (B) (80 µg each)
activated by 100 µM forskolin and 0.24 µM
G s was performed in the indicated peptide concentration.
The specific activities of mAC7 and mAC6 were 17 and 8 nmol·min
1·mg
1, respectively. The
means ± S.E. are representative of at least three
experiments.
-D-galactopyranoside (X-gal)
for yeast strain EGY48(pSH18-34) that contained 7C1a or 7C2a fused to
the DNA binding domain of LexA (LexA-DB-7C1a or LexA-DB-7C2a,
respectively) (data not shown). When the 7C1b-B42AD fusion was
coexpressed in yeast strains containing LexA-DB-7C1a or LexA-DB-7C2a, a
>2-fold increase was observed in the
-galactosidase activity
(standard
-galactosidase unit multiplied by 1000: 7C1a with control
plasmid = 36 ± 1 while 7C1a with 7C1b = 84.6 ± 0.4 and 7C2a with control = 40 ± 2 while 7C2a + 7C1b = 85.8 ± 0.5). This finding suggests that 7C1b can interact with
both 7C1a and 7C2a.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s. Instead, catalysis is activated by an induced
juxtaposition of these two domains to properly form the catalytic cleft
at their interface. Interestingly, the structure of 2C2 homodimer and
5C1a/2C2a heterodimer reveals significant contact between C1a and C2a
(23, 26). Because C1a and C2a are tethered by the transmembrane domains
(M1 and M2) in their native membrane-bound configuration, the question
arises of how C1a and C2a are kept in an inactive conformation in the
resting state of the enzyme.
s complex can be coimmunoprecipitated.2 Interestingly,
7C1b-SlyD can compete with G
s to bind the 7C1a-7C2a complex, suggesting that one possible mechanism for 7C1b-mediated inhibition is blocking the binding of G
s to 7C1a-7C2a
complex.2 However, the presence of SlyD in our 7C1b
preparation precludes drawing any definitive conclusion. It would be
interesting to determine whether 7C1b directly interacts with 7C1a and
7C2a and whether the interaction of 7C1b to 7C1a and 7C2a is involved
in 7C1b-mediated inhibition. It is worth noting that Scholich et al. (20) have shown that AC5 C1b can interact with 5C2a by yeast two-hybrid analysis. Unfortunately, they did not test the interaction of AC5 C1b with its C1a domain.
i in AC5 and
regulation by calcineurin in AC9 (17, 19, 42). We show here that
peptides from the C1b region of different isoforms of adenylyl cyclase
can inhibit the activity of a subset of adenylyl cyclase isoforms. This
isoform-specific inhibition by peptides may provide a tool with which
to address the physiological functions of each isoform of adenylyl
cyclase in the intact cells (43).
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ACKNOWLEDGEMENTS |
---|
We are grateful for the help in oligonucleotide synthesis and DNA sequencing from P. Gardner (Howard Hughes Medical Institute, University of Chicago), protein sequencing from Andrew Bohm (Boston Biomedical Research Institute), plasmids for the expression of SlyD and its mutants from Bill Roof (Texas A&M University), and insightful suggestions from R. Iyengar (Mount Sinai School of Medicine) and Ryland F. Young (Texas A&M University).
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FOOTNOTES |
---|
* This research was supported by National Institute of Health Grant GM53459 and American Heart Association Established Investigator Award (to W. -J. T.), National Institute of Health Grant DK38761 (to R. Iyengar), and Fellowship from American Heart Association Chicago Affiliate and University of Chicago Committee on Cancer Biology (to S. -Z. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Neurobiology, Pharmacology, and Physiology, The University of Chicago, 947 E. 58th St., MC0926, Chicago, IL 60637. Tel.: 773-702-4331; Fax: 773-702-3774; E-mail: wtang@uchicago.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M010361200
2 S.-Z. Yan, J. Beeler, and W.-J. Tang, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
Gs, subunit
of the G protein that stimulates adenylyl cyclase;
AC, adenylyl cyclase;
PPIase, cis-trans
peptidylprolyl isomerase;
SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis;
NI-NTA, nickel-nitrilotriacetic acid.
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