(Received for publication, February 2, 1996; and in revised form, March 14, 1996)
From the
Mammalian adenylyl cyclases have two homologous cytoplasmic
domains (C and C
). The first cytoplasmic domain
of type I enzyme (IC
) and the second cytoplasmic domain of
type II enzyme (IIC
-
3, a construct in which 36
N-terminal amino acids of the C
region are deleted) were
expressed and purified to homogeneity. Alone, each had no adenylyl
cyclase activity; however, mixing of the two domains in vitro resulted in G
- and forskolin-activated enzyme
activity. The turnover number for G
- and
forskolin-stimulated enzyme activity of the complex between IC
and IIC
-
3 was 8.2 s
. The
concentration of IIC
-
3 to achieve half-maximal
activation of IC
was 0.8 and 1.3 µM when
stimulated by forskolin and G
, respectively. The
concentration of IIC
-
3 needed to complex with IC
was reduced 10-fold (0.08 µM) when the enzyme was
activated by both forskolin and G
, suggesting that
G
and forskolin increased the affinity of the two
cytoplasmic domains for each other.
The enzymatic activity of adenylyl cyclase is the key step in
regulating the intracellular cAMP concentration upon stimulation of a
variety of hormones, neurotransmitters, and other regulatory molecules.
There are at least nine distinct mammalian adenylyl cyclases which have
a similar structure (Fig. 1A)(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
This includes two intensely hydrophobic domains (M and
M
) and two
40-kDa cytoplasmic domains (C
and C
). The C
and C
domains
contain sequences (C
and C
) that are similar
to each other and to other adenylyl and guanylyl
cyclases(12, 13) . Each isoform of adenylyl cyclase
has its own distinct tissue distribution and unique regulatory
properties, providing modes for different cells to respond diversely to
similar stimuli(12, 14) .
Figure 1:
Properties of soluble type
I-type II adenylyl cyclase constructs expressed in E. coli. A, the top shows a model of mammalian adenylyl
cyclase with each of its regions labeled. ACI = type I
enzyme; ACII = type II enzyme. Below are shown the
constructs used in this work. The first four include, at their amino
termini, a hexa-histidine and a HA1 epitope tag with a short linker to
create the EcoRI and NcoI restriction sites. B, adenylyl cyclase activities and protein expression of the
ICIIC
construct in protease-deficient E.
coli strains, BL21DE3 and SG22094. Samples (10 µl) were taken
for enzyme assay and immunoblot assay with either 12CA5 or C2-1077
antibodies at the indicated times after IPTG induction. C,
adenylyl cyclase activities for a mixture of 10 µl of a bacterial
lysate (from BL21DE3 cells) containing the components at the top that
were obtained after the indicated hours of induction with IPTG and 10
µl of an lysate (from BL21-DE3 cells) containing either IIC
or IC
. The immunoblot shows the amount of the
component at the top after the indicated hours of induction by IPTG. p.i. = post-IPTG induction. Adenylyl cyclase activity
is shown as
nmol
min
mg
. Data are
representative of two experiments.
Membrane-bound adenylyl
cyclases are expressed in small quantities, and the enzyme is labile
and difficult to manipulate in detergent-containing solutions. To
facilitate biochemical and structural analysis, a soluble adenylyl
cyclase has been constructed by linking the C and C
domains of type I and type II adenylyl cyclases,
respectively(15) . The resulting protein is sensitive to
activation by G
(
)and forskolin and to
inhibition by P-site inhibitors, indicating the essential roles of
C
and C
domains for catalysis and regulation.
In this paper, we describe the expression and purification of the
C
and C
domains of type I and type II
adenylyl cyclase, respectively. Alone, each has no adenylyl cyclase
activity; however, mixing of the two domains in vitro results
in G
- and forskolin-activated enzyme activity.
Figure 5:
Superdex 200 gel filtration chromatography
of purified IC, IIC
-
3, mixed
IC
and IIC
-
3, and an extract containing
IC
IIC
using
T
E
D
N
(A)
and T
D
N
with 10 mM MgCl
, 1 mM ATP, and forskolin (B).
Molecular size markers (Bio-Rad) are thyroglobin (670 kDa),
-globulin (158 kDa), chicken ovalbumin (44 kDa), and horse
myoglobin (17 kDa). A, total activity values of the purified
IC
(5 µg), IIC
-
3 (5 µg), mixed
IC
(0.5 µg) and IIC
-
3 (5 µg), and
an extract containing IC
IIC
(300 µg) were
80, 183, 47, and 9.9 nmol
min
, respectively. B, total activity values of the purified IC
(5
µg), IIC
-
3 (5 µg), mixed IC
(1
µg) and IIC
-
3 (50 µg), and an extract
containing IC
IIC
(300 µg) were 82.5, 66.2,
115.8, and 6.37 nmol
min
, respectively. Data
are representative of two experiments.
A 30-kDa proteolytic fragment of
ICIIC
was detected using antiserum C2-1077,
suggesting that there is a prominent cleavage at the junction between
IC
and IIC
. To investigate whether the complex
of IC
and IIC
was part of a catalytically
active species of the proteolyzed IC
IIC
, HA1
and hexo-histidine-tagged IC
and IIC
were
expressed separately. Using the monoclonal antibody from 12CA5, the 30-
and 31-kDa proteins were detected in high speed supernatants of lysates
from E. coli that expressed IC
or IIC
(expected molecular mass as 27 and 31 kDa, respectively),
indicating that the IC
and IIC
protein were
stable, soluble proteins. Adenylyl cyclase activities of E. coli lysates that expressed either IC
or IIC
were not different from those of lysates of E. coli that
carried the control vector (
0.01
nmol
min
mg
). However,
mixing of the lysates, each expressing one of these constructs,
resulted in high enzyme activity (2-9
nmol
min
mg
, Fig. 1C). The enzyme activity correlated generally with
expression (monitored by immunoblot) of IC
and IIC
from cells (Fig. 1C).
Figure 2:
Purification of IC and
IIC
-
3. A, purification on a Ni-NTA column.
Lysates of E. coli that expressed IC
,
IIC
, and IIC
-
3 (1.5 ml) were passed
through a 0.3-ml Ni-NTA column, and proteins that flowed through the
column were collected. The column was subsequently washed with 1.5 ml
of wash 1 buffer,
T
N
P
, and that
of wash 2 buffer,
T
N
I
P
.
The column was then eluted with 1.5 ml of
T
N
I
P
. Lane 1, load; lane 2, flow-through; lane 3,
wash 1; lane 4, wash 2; and lane 5, eluate. Total
adenylyl cyclase activities (nmol
min
) of
IC
, IIC
, and IIC
-
3 lysates
were 72, 38, and 75, respectively. Activities of flow-throughs, washes,
and eluates are given as a percentage of adenylyl cyclase activity
applied. Immunoblot was performed with antibody, 12CA5. Data are
representative of two experiments. B, Coomassie Blue stain of
purified IC
(0.2 µg) and IIC
-
3 (5
µg). C, immunoblot of purified IC
and
IIC
-
3 (100 ng each).
The same procedure did not succeed in the
purification of IIC. The majority of the adenylyl cyclase
activity from IIC
did not bind to Ni-NTA, probably due to
proteolysis or masking of the hexo-histidine tag. When lysates
containing IC
were mixed with lysates containing IIC
and applied to Ni-NTA column, most of adenylyl cyclase did not
bind to the column (data not shown). This indicated that the binding
between IC
and IIC
was not strong enough for
copurification of IC
and IIC
.
To purify
IIC, we used IIC
-
3, a construct that
deleted 36 N-terminal amino acids of IIC
, residues that are
not conserved among mammalian adenylyl cyclases. HA1 and
hexo-histidine-tagged IIC
-
3 protein was expressed as a
soluble protein, based on immunoblot, and formed G
-
and forskolin-regulated adenylyl cyclase when mixed with lysate
containing IC
in vitro (Fig. 1C).
IIC
-
3 could be purified by Ni-NTA and, after
subsequent chromatography on FPLC-Mono Q, 95% pure protein (29 kDa) was
obtained (Fig. 2B). Its identity was confirmed by
immunoblot (Fig. 2C). The recovery of adenylyl cyclase
activity was about 35%, and the yields for IIC
-
3
proteins were 2 mg from each liter of E. coli culture.
Figure 3:
Enzyme activity of a mixture of IC and IIC
-
3 (0.2 µg each). A,
activation by forskolin; B, activation by
G
-GTP
S; C, synergistic activation by
G
-GTP
S and forskolin; D, inhibition by
2`-deoxy-3`-AMP. The concentration of GTP
S-G
is
200 nM in C. Sum (Fsk+G
) is the sum of adenylyl cyclase
activities observed in the presence of forskolin or
GTP
S-G
alone; Fsk + G
is
adenylyl cyclase activity observed in the presence of both
GTP
S-G
and forskolin. The means ± S.E. are
representative of two experiments.
Increased
concentrations of IIC-
3 markedly increased adenylyl
cyclase activity when added to a fixed amount of IC
(6
nM) (Fig. 4A). The half-saturable
concentration (EC
) of IIC
-
3 for
forskolin- and G
-GTP
S-activated activity was 0.8
and 1.3 µM, respectively. When G
and
forskolin were used together, EC
of IIC
-
3
fell about 10-fold to 0.08 µM. This suggested that the
synergistic effects of G
-GTP
S and forskolin on
enzyme activity reflected an increase in the affinity of IC
and IIC
-
3 for each other.
Figure 4:
Complementation of adenylyl cyclase
activity. A, complementation of IC by
IIC
-
3. B, complementation of
IM
C
-(1-570) or
IM
C
-(1-484) by IIC
-
3. C, complementation of IM
C
or
IIM
C
by IC
. Purified IC
(20 ng), or Sf9 cell membranes (20 µg) containing
IM
C
-(1-570) or
IM
C
-(1-484) were mixed with the indicated
quantities of IIC
-
3 on ice for 10 min before the
assay. Sf9 cell membranes containing IM
C
or
IIM
C
(20 µg) were mixed with the indicated
quantities of IC
. Adenylyl cyclase assays were performed at
30 °C in the presence of 100 µM forskolin (A, Fsk, B, and C) or 100 µM forskolin and 200 nM GTP
S-G
(A, Fsk+G
). The means
± S.E. are representative of two
experiments.
Purified IC or IIC
-
3 were subjected to gel filtration on
Pharmacia Superdex 200 using
T
E
D
N
as the buffer.
A major peak of adenylyl cyclase activity consistent with a globular
30-kDa protein was observed, half of the size for the enzyme activity
of lysates containing IC
IIC
(Fig. 5A). The molecular size did not shift for
the mixture of IC
and IIC
-
3, presumably
due to the low affinity between two molecules (Fig. 5A).
To investigate whether a complex of
IC and IIC
-
3 could be detected, gel
filtration was performed in the presence of forskolin under the
conditions for the enzyme assay (forskolin, 1 mM ATP, and 10
mM MgCl
and the minimal concentration (100
mM) of NaCl; Fig. 5B). A major peak of
adenylyl cyclase activity consistent with a globular 30-kDa protein was
observed when the purified IIC
-
3 was applied alone,
half of the size for the enzyme activity of lysates containing
IC
IIC
. When IC
alone was applied, a
major peak of adenylyl cyclase activity consistent with a globular 55
kDa was observed; the shift from 30- to 55-kDa protein was due to the
lower concentration of NaCl. This suggested that IC
might
exist as a dimer or as a nonglobular protein at lower salt
concentrations (100 mM). When the mixture of IC
(1
µg) and IIC
-
3 (50 µg) was tested, a peak of
adenylyl cyclase activity consistent with 45-kDa proteins was observed.
The shift in elution profile suggested that IC
and
IIC
-
3 did interact. The low apparent molecular mass
(45 kDa instead of the expected 60 kDa) could be accounted for by
dissociation of the complex of IC
and
IIC
-
3 and/or an unusual shape of the complex.
There has been
considerable speculation about the roles of the transmembrane domains
of adenylyl cyclases(12) . The transmembrane domains target
adenylyl cyclase to the plasma membrane for interaction with, and
thereby regulation by, G proteins. Our studies indicate that the two
cytoplasmic domains of mammalian adenylyl cyclases do not appear to
have high affinity for each other. EC for IIC
to complex with IC
is 0.8 and 1.3 µM in
forskolin- and G
-stimulated activity, respectively.
Affinity between two natural linked cytoplasmic domains (IC and IC
) is at least 10-fold less than that between
IC
and IIC
. Thus, the transmembrane domain
(M
) could link and facilitate the interaction of the two
cytoplasmic domains by creating a high local concentration. It remains
to be determined whether the transmembrane domains have additional
functions, such as altering the interaction between two cytoplasmic
domains for regulations or serving as pore structures.