(Received for publication, May 9, 1997)
From the Department of Pharmacological and
Physiological Sciences, University of Chicago, Chicago, Illinois 60637 and the § Laboratory of Molecular Biology, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892-0580
The interaction between the subunit of G
protein Gs (Gs
) and the two
cytoplasmic domains of adenylyl cyclase (C1 and C2) is a key step in the stimulation of cAMP synthesis by
hormones. Mutational analysis reveals that three discrete regions in
the primary sequence of adenylyl cyclase affect the EC50
values for Gs
activation and thus are the affinity
determinants of Gs
. Based on the three-dimensional
structure of C2·forskolin dimer, these three regions
(C2
2, C2
3/
4, and C1
1) are close together and form a negatively charged and hydrophobic
groove the width of an
helix that can accommodate the positively
charged adenylyl cyclase binding region of Gs
. Two
mutations in the C2
3/
4 region decrease the
Vmax values of Gs
activation
without an increase in the EC50 values. Since these three
regions are distal to the catalytic site, the likely mechanism for
Gs
activation is to modulate the structure of the active
site by controlling the orientation of the C2
2 and
3/
4 structures.
Mammalian adenylyl cyclase is the enzyme responsible for
integrating multiple extracellular and intracellular signals to
generate cAMP and thus activate cAMP-dependent protein
kinase and cyclic nucleotide-gated ion channels (1, 2). All nine cloned
mammalian and Drosophila rutabaga adenylyl cyclases are
stimulated directly by the subunit of Gs
(Gs
),1 and all but type IX
are activated by forskolin. Gs
and forskolin bind and
activate adenylyl cyclases separately or synergistically when presented
together (3, 4). Mammalian adenylyl cyclases are integral membrane
proteins consisting of two homologous cytoplasmic domains
(C1 and C2), each following a membrane domain
(M1 and M2) (1, 2). The C1 and
C2 domains form the catalytic core and can be engineered as
a Gs
- and forskolin-sensitive soluble adenylyl cyclase,
i.e. by mixing of IC1 protein (C1
domain of type I adenylyl cyclase) and IIC2 protein
(C2 domain of type II adenylyl cyclase) in vitro
(5-8). In this paper, we describe mutations at three discrete regions
of the soluble adenylyl cyclase, one in the IC1 protein and
two in the IIC2 protein, that significantly affect
Gs
activation with little change in forskolin
activation.
Plasmids used to express mutant forms of IC1 and IIC2 were constructed by site-directed mutagenesis using pProExHAH6-IC1 or -IIC2 as the phagemid (9). Oligonucleotides used for mutagenesis contained 10-12 complementary nucleotides flanking each side of the target codon(s) that was replaced with the appropriate codon. Mutations were confirmed by dideoxy nucleotide sequencing of phagemid DNA.
To express wild type and mutant forms of hexohistidine-tagged
IC1 and IIC2, the plasmids that encoded wild
type or mutant forms of IC1 or IIC2 were
transformed into Escherichia coli BL21(DE3) cells. E. coli cells that harbored the desired plasmid were cultured in T7
medium containing 50 mg/ml ampicillin at 30 °C (10). When A600 reached 0.4, isopropyl-1-thio--D-galactopyranoside (100 µM) was added. After 3-4 h, the induced cells were then
collected and lysed; IIC2 proteins were purified using the
nickel nitrilotriacetic acid column and fast protein liquid
chromatography Q-Sepharose column as described (5). The Coomassie Blue
staining of SDS-polyacrylamide gel electrophoresis was used to
determine the protein peak in the fractions from Q-Sepharose column.
The concentration of proteins was determined using Bradford reagent and
bovine serum albumin as standard (11). The construction of plasmid
H6-pQE60-Gs
and the expression and
purification of hexohistidine-tagged Gs
were performed
as described (10). Gs
was activated by 30 µM AlCl3 and 10 mM NaF, and
adenylyl cyclase assays were performed at 30 °C for 20 min (5,
12).
The Gs structure
was modeled using the sequence alignment and homology-modeling program
LOOK version 2.0 (Molecular Applications Group) based on its sequence
homology to GTP
S-bound forms of bovine G protein transducin
(13). The same protocol was tested by modeling the structure of
Gi
, which resulted in a model closely agreeing with
GTP
S-bound Gi
structure (the root mean square
deviation of the C
atoms was found to be 1.17 Å) (14). A
C1C2 heterodimer was modeled based on the
structure of (IIC2)2·forskolin2
(15). Gs
was docked onto the
C1C2 heterodimer using program O (16) and data
from the mutational analysis of Gs
and
C1C2 soluble adenylyl cyclase (Ref. 17 and this
paper).
We use the sequence
comparison to guide the mutagenic mapping of the Gs
binding site (Fig. 1). The IIC2, but not the IC1, protein has weak Gs
- and
forskolin-stimulated activity (~1000-fold less than mixed
IC1 and IIC2 proteins) (18). Thus, the
C2 domain must include amino acid residues that contribute to binding and partial activation by Gs
and forskolin.
Some of these residues are expected to be conserved among the
C2 domain of mammalian and fly adenylyl cyclases but might
not be conserved among the C1 domain of mammalian and fly
adenylyl cyclases and the cyclase domains of membrane-bound guanylyl
cyclases. Fourteen IIC2 mutants (to either alanine or
leucine) at the 13 residues that fit this criterion were constructed,
and all of them had relatively normal expression based on immunoblot
(Figs. 1 and 2 (mutants IIC2
C911A, R913A, I919A, and D921A
and 10 other mutants not shown). We then tested for Gs
and forskolin activation using E. coli lysates containing
the IIC2 mutant proteins and wild type IC1
protein (Table I). Due to the semiquantitative nature of
using E. coli lysates, we graded the enzyme activity of the
lysates containing IIC2 mutants relative to that containing wild type IIC2 as follows: near normal (+++, >50% of the
control), moderately reduced (++, 25-50% of the control),
significantly reduced (+, 5-25% of the control), and little or no
activation (±, <5% of the control). We expected that mutations at
the Gs
binding site in the IIC2 protein
would cause a significant reduction in Gs
activation but
have little or no effect in forskolin activation. Only two of these
IIC2 mutants, R913A and D921A, fit these
criteria.2
|
To confirm that IIC2 mutants R913A and D921A had
reduced Gs activation and to further characterize these
mutants, we purified both IIC2 R913A and D921A to
homogeneity and tested for their Gs
- and
forskolin-activated activity when mixed with purified IC1
protein in vitro (Figs. 2 and 3; Table I).
Both IIC2 R913A and D921A had near normal enzyme activity
when stimulated by forskolin, whereas they had about a 15-fold
reduction in Gs
-stimulated activity (Fig. 3,
A and B; Table I). In the presence of 10 µM forskolin, both IIC2 R913A and D921A had
relatively normal Vmax values but had
significantly increased EC50 values for Gs
activation (Fig. 3C and Table I).
While this research was in progress, the three-dimensional structure of
the IIC2·forskolin complex was solved, and the structure revealed that Arg-913 and Asp-921 were located on the amphipathic 2
helix (Fig. 1) (15). To test the effect of mutations at the conserved
residues located at the hydrophilic surface of
2, IIC2 mutants E910A, L914A, N916A, E917A, and D924A were constructed and
tested for their activity in response to Gs
and
forskolin activation (Table I; Figs. 2 and 3). Similar to
IIC2 mutants R913A and D921A, the lysates containing
IIC2 E910A, L914A, N916A, and E917A had significantly
reduced or little Gs
activation but only moderate
reduction in forskolin activation (Table I). The D924A mutation did not
affect Gs
and forskolin activation (data not shown).
IIC2 E910A, L914A, N916A, and E917A were purified to
homogeneity and tested for their activation by Gs
and forskolin (Figs. 2 and 3; Table I). All four mutants had about a
10-fold reduction in Gs
activation and less than a
2-fold reduction in forskolin activation. Similar to IIC2
R913A and D921A, all four mutants had significant increases in
EC50 values for Gs
activation. These data
indicate that six amino acid residues, Glu-910, Arg-913, Leu-914,
Asn-916, Glu-917, and Asp-921, of IIC2 are involved in
Gs
activation.
In
contrast to the sensitivity of other mammalian and fly adenylyl
cyclases to both Gs and forskolin, type IX enzyme is
activated by Gs
but not by forskolin (19). We
hypothesize that the crucial residue(s) for forskolin binding is
missing in the C2 domain of type IX enzyme. Sequence
comparison among the C2 domains reveals that eight amino
acid residues are absolutely conserved among type I-VIII and rutabaga
adenylyl cyclases but differ in type IX enzyme (Fig. 1). Five of them
(Gln-880, Ser-881, Ser-942, Ser-990, and Asn-992) have been mutated to
alanine and tested for their activation by Gs
and
forskolin.3 Fortuitously, another region
that affects Gs
activation was revealed. Lysates
containing mutant IIC2 N992A had near normal forskolin
activation but a significantly reduced Gs
activation. Lysates containing mutant IIC2 S990A had near normal
Gs
and forskolin activation; however, a consistent
2-fold higher relative percent of Gs
activation
(119 ± 13%) than of forskolin activation (57 ± 10%) was
observed. When we tested a lysate containing the IIC2
double mutant S990A/N992A, the Gs
-and
forskolin-activated activity was near normal, and the percent of
Gs
activation (76 ± 9%) was less than that of
forskolin activation (139 ± 28%).
To further characterize IIC2 S990A, N992A, and S990A/N992A,
the three mutant proteins were purified to homogeneity and tested for
Gs and forskolin activation (Figs. 2 and
4; Table I). IIC2 S990A was normal in
forskolin activation, whereas IIC2 N992A and S990A/N992A
had only a slight reduction in forskolin activation (Table I and Fig.
4A). Interestingly, IIC2 S990A had about
3-fold-enhanced Gs
activation, whereas IIC2
N992A had 4-fold-reduced Gs
stimulation (Table I and
Fig. 4B). The Gs
activation of double mutant
IIC2 S990A/N992A was nearly normal, presumably due to
compensation by the two mutations (Table I and Fig. 4B).
When simultaneously stimulated by Gs
and forskolin,
IIC2 N992A had a lower Vmax value
but relatively normal EC50 value (Table I and Fig.
4C). In contrast, IIC2 S990A had a decrease in
both EC50 and Vmax values; the
decrease in EC50 could explain the apparent higher
Gs
activation when assayed only with Gs
(Table I and Fig. 4C). Double mutant IIC2
S990A/N992A had a near normal Vmax value and a
slightly increased EC50 value. The three-dimensional structure of IIC2·forskolin reveals that Ser-990 is the
only residue that joins the
3 and
4 regions of IIC2;
thus, it might play a pivotal role in controlling the relative
orientation between
3 and
4 of IIC2 (Fig. 1). How the
change from Ser-990 to Ala alters both EC50 and
Vmax values for Gs
activation
remains elusive.
To further examine the region containing Ser-990 and Asn-992, we
constructed and tested six more alanine-scanning IIC2 point mutants in the Asn-987-Lys-995 region. Two more amino acid residues, His-989 and Phe-991 were shown to be involved in Gs
activation.4 Lysates containing
IIC2 H989A and F991A had a significantly reduced Gs
activation but had near normal or moderately reduced
forskolin stimulation, respectively (Table I). Similar results were
observed when the purified mutant proteins were used (Table I and Fig. 4). When the mutants were stimulated by Gs
and forskolin simultaneously, IIC2 H989A exhibited a lower
Vmax value but relatively normal
EC50 value. When the same assay was applied to
IIC2 F991A, a significant increase in EC50
value was observed; due to low enzyme activity, the
Vmax value of this mutant could not be
determined. It is worth noting that two IIC2 mutants in
this region (at the
3/
4 region, IIC2 S990A and N992A)
had reductions in Vmax values but had little
increases in EC50 values (Fig. 4C); this is in contrast to the IIC2
2 mutants that all have increased
EC50 values.
The C1 and
C2 domains of mammalian adenylyl cyclase have ~25-50%
identity, and there is a high degree of sequence conservation between
dimer interface residues in C1 and C2 based on
the interaction of the IIC2 dimer in the
(IIC2)2·forskolin2 crystal
structure (15). Thus, the interaction between C1 and
C2 domains might be similar to that of the IIC2
dimer in (IIC2)2·forskolin2
crystal structure. Since the 2 region of the IIC2
protein is close to the interface of the IIC2 dimer, we
asked whether Gs
could interact with the amino acid
residue(s) located at the C1 domain near the
2 helix of
the IIC2 protein in order to facilitate the interaction
between the C1 and C2 domains. The contact of
the IIC2 dimer in IIC2·forskolin model
predicts that the sequences at the proposed N terminus of
IC1 are likely candidates (Fig. 6A). Truncation
analysis revealed that the IC1 mutant,
271-292, a
deletion of amino acid residues 271-292, had normal Gs
or forskolin activation (Table I). We then constructed and tested the
Gs
- and forskolin-stimulated activity of four
IC1 mutants, F293A, H294A, S305A, and L307A, that have a
mutation in the N-terminal region of IC1. Only one
IC1 mutant, IC1 F293A, exhibited little Gs
activation but retained a near normal forskolin
stimulation when either E. coli lysate containing
IC1 F293A or purified IC1 mutant protein (Figs.
2 and 5; Table I) were
used.5 When stimulated by Gs
and forskolin, a significant increase in the EC50 value of
mutant IC1 F293A was also observed (Fig. 3). We also tested
the conserved amino acid residues in the putative
4/
5 region,
which is adjacent to the putative N terminus of IC1, and
found that none of the mutants exhibited a preferen-tial reduction in
Gs
activation.6 These data
indicate that the conserved Phe-293 at the C1 domain is
crucial in Gs
activation. In addition, the data provide support for the idea that the structure of the C2 dimer is
valid in examining the structure of the C1C2
heterodimer.
A, schematic of C1
(green)C2 (white) model based on
(IIC2)2·forskolin2 crystal
structure illustrating the binding sites for Gs
(red) and G
(yellow) (24). Forskolin is
shown with white bonds where it binds at either end of the
active site cleft. B, space-filling model of
Gs
·adenylyl cyclase complex. The upper
panel shows the docked view, and the lower panel shows two molecules rotated by 90 degrees. Residues of both Gs
(17) and adenylyl cyclase (this paper) implicated in binding are
highlighted in red. The structure of
Gs
was modeled based on the crystal structure of
GTP
S-bound G protein transducin
(13). The membrane surface is
based on the crystal structure of the complexes of both
Gi/t
chimera·G
and Gi
·G
,
and it is at least 28 Å away from plasma membrane (25, 26). The
structure of the C1C2 heterodimer was modeled
on the basis of the crystal structure of
(IIC2)2·forskolin2 (15).
N-C1, N terminus of C1. C,
surface representation of adenylyl cyclase and Gs
. The coloring is according to electrostatic potential using GRASP (27) and contoured in the range from
5kT
(red) to +5kT (blue). The figure shows the
complementary nature of the interaction surfaces, negative on the
adenylyl cyclase and positive on Gs
. The orientation
matches the lower panel of B.
The mutagenesis based on the sequence comparison of adenylyl and
guanylyl cyclases and the molecular structure of IIC2 has revealed that 10 amino acid residues (Glu-910, Arg-913, Leu-914, Asn-916, Glu-917, Asp-921, His-989, Ser-990, Phe-991, and Asn-992) within two regions (2 and
3/
4) of IIC2 are
essential for Gs
activation. Although these two regions
of IIC2 proteins are 68 amino acids apart in the primary
sequence, they are in close proximity in the structure of the
IIC2·forskolin dimer (Fig. 6, A
and B). The Gs
binding site is separate from
but close to the proposed G protein
binding site (N terminus of
3) of type II adenylyl cyclase, which is consistent with ability of
the G
to synergize the Gs
activation of type II
enzyme (Fig. 6A) (20-22). The putative Gs
binding site forms a negatively charged and hydrophobic groove 10 × 10 × 15 Å, capacious enough to bind an
helix (Fig. 6,
B and C).7 This
negatively charged groove could attract the positively charged surface
formed by the putative adenylyl cyclase binding region of
Gs
(
2/
4 (switch 2),
3/
5, and
4/
6) (13,
14, 17) (Fig. 6, B and C). It is also worth
noting that the sequences at the C1
2 region are
reasonably conserved among the Gi
-sensitive adenylyl
cyclase (types I, V, and VI) but not other isoforms; thus, it may be
the determinant for Gi
binding, a site independent of
that for Gs
(23).
The complex of C1 and C2 domains are necessary
for potent activation by Gs. Although how the
C1 domain interacts with the C2 domain remain
elusive, we hypothesize that their interaction is similar to the
contact of the IIC2 dimer based on the following observations: the relative high degree homology of C1 and
C2 domains, the sequence conservation at the dimer
interface of C1 and C2 domains based on the
structure of IIC2 dimer, and the success of the
C1C2 model to predict the importance of the N
terminus of C1 domain for Gs
activation. The
C1C2 model was constructed using the homology
modeling based on the (IIC2·forskolin)2
structure (Fig. 6). The model shows that the putative adenylyl cyclase
binding regions of Gs
can dock well to the negatively
charged groove that is its presumed site in adenylyl cyclase (Fig.
6B); the validity of this model remains to be tested
experimentally.
How does Gs activate adenylyl cyclase? Based on
mutational analysis, we hypothesize the following events leading to the activation of adenylyl cyclase by Gs
. The greatest
effects on EC50 values for Gs
activation map
to the C2
2 helix, suggesting that in the first step,
Gs
binds to adenylyl cyclase with an energetic driving
force provided primarily by the
2 helix of the C2
region. The sensitivity to mutation at Phe-293 demonstrates a potential
role for Gs
in bridging the C1 and
C2 domains and promoting their juxtaposition in a
catalytically productive manner. The observation that mutation of the
3/
4 of C2 can alter the Vmax
for Gs
-stimulated catalysis suggests that this region is
an allosteric linker between the Gs
binding site and the active site. Indeed, both
2 and
4 directly participate in forming the ventral cleft-containing active site. The Gs
binding regions occur on portions of the two longest
helices in the cyclase
structure. The
2 and
3 helices provide 30- and 37-Å-long lever
arms, respectively, such that a modest change in their mutual orientation at the Gs
binding site could be converted
scissorswise into a large change in the structure of the active site,
leading to catalytic activation. The determination of the molecular
structure of C1C2 and
Gs
C1C2 is in progress, and the
solution will yield valuable insight into how Gs
activates adenylyl cyclase.
We thank P. Gardner at Howard Hughes Medical Institute for speedy oligonucleotide synthesis and W. Epstein, A. Bohm, P. B. Sigler, M. Villereal, and C. Drum for the critical reading of this manuscript.