From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0636
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ABSTRACT |
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We describe the development of a genetic system
allowing for the isolation of mutant mammalian adenylyl cyclases
defective in their responses to G protein subunits, thus allowing for
the identification of structural elements within the cyclase that are
responsible for the recognition of these regulators. Expression of
mammalian type V adenylyl cyclase in a cyclase-deleted yeast strain can
conditionally complement the lethal phenotype of this strain. Type V
adenylyl cyclase-expressing yeast grow only when the cyclase is
activated by coexpression of Gs or addition of
forskolin to the medium; however, growth arrest is observed in the
presence of both activators or under basal conditions. Utilizing this
genetic system, we have isolated 25 adenylyl cyclase mutants defective
in their response to Gs
. Sequence analysis and
biochemical characterization of these mutants have identified residues
in both cytoplasmic domains of the cyclase that are involved in the
specific binding of and regulation by Gs
.
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INTRODUCTION |
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Regulation of intracellular cyclic AMP concentrations is
principally controlled at the level of its synthesis, through the hormonal regulation of adenylyl cyclase, the enzyme responsible for the
conversion of ATP into cyclic AMP. The adenylyl cyclase system
comprises three components: seven transmembrane-spanning receptors for
a variety of hormones and neurotransmitters, heterotrimeric G
proteins,1 and the catalytic
entity itself. Currently, nine isoforms of membrane-bound adenylyl
cyclases have been identified by molecular genetic approaches, and
studies of these enzymes reveal both common and unique regulatory
features (1, 2). All isoforms tested to date are activated by the
GTP-bound form of Gs and by forskolin; for some of the
isoforms, such as the type V, these stimulators synergistically
activate the enzyme. All isoforms of adenylyl cyclase are further
regulated by additional inputs in an isoform-specific pattern. For
example, Gi
inhibits the types I, V, and VI isoforms
(3-5);
subunits can activate (types II, IV, and VII) or inhibit
(type I) adenylyl cyclase activity (6-10). Increases in intracellular
calcium concentrations will inhibit the types V and VI isoforms
(11-13) while indirectly activating (via a
calmodulin-dependent process) types I and VIII (14, 15) and
inhibiting (by calmodulin kinase) the type III isoform (16).
Structural motifs responsible for the recognition of these regulatory
molecules by the adenylyl cyclases are starting to be uncovered. A
region of type II adenylyl cyclase (residues 956-982 from the
C2 domain) containing a QXXER motif has been
shown to be important for the regulation of this enzyme by G protein
subunits (17). This site has been proposed to interact with the
subunit of the G protein, within the amino-terminal 100 residues
of
(18, 19). Synthetic peptide and mutational approaches have
identified sequences located within the first cytoplasmic (C1a) region of type I adenylyl cyclase (residues 495-522)
important for calmodulin activation (20, 21); this sequence has a
hydrophobic/basic composition and an aromatic amino acid in its
NH2-terminal portion, typical of most calmodulin binding
domains, and is therefore likely to function as the calmodulin binding
domain in the cyclase. The recently solved crystal structure of a
homodimer of the type II adenylyl cyclase second cytoplasmic domain
(C2) bound with forskolin has revealed the binding site(s)
of this regulator (22). Mutational analysis of Gs
has
revealed three regions of the protein critical for the ability of
Gs
to activate adenylyl cyclase (23, 24); however, at
the time that this study was initiated, the Gs
-binding
site on the adenylyl cyclase molecule had not been determined.
The yeast adenylyl cyclase, encoded by the CYR1 gene, is
structurally distinct from and exhibits much different regulation than
the mammalian adenylyl cyclases (25). Yeast strains containing a
disruption of the CYR1 gene are not viable but can be
propagated in medium containing cAMP, demonstrating the importance of
cAMP for growth of the organism (26). Based on this requirement of cAMP
for growth, we have expressed mammalian adenylyl cyclases in a
CYR1-deleted yeast strain to take advantage of the readily manipulatable genetic properties of the yeast for the isolation of
mutant mammalian adenylyl cyclases defective in their regulatory responses. In this report, we describe a genetic system utilizing the
expression of mammalian type V adenylyl cyclase in yeast and demonstrate its utility in isolating mutant adenylyl cyclases defective
in their regulation by Gs. Genetic and biochemical analyses of these mutants reveal structural information about the
Gs
binding site on the cyclase, as well as regions of the molecule important for responses to activating regulators.
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EXPERIMENTAL PROCEDURES |
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Yeast Strains and Plasmid Constructions--
Yeast strain TC41-1
(27) (MAT a, leu2-3, leu2-112, ura3-52, his3, his4, cam1,
cam2, cam3, cyr1 ::URA3) was a generous
gift of Warren Heideman (University of Wisconsin, Madison, WI). Strain 12229 is an isogenic derivative of TC41-1 generated by the targeted integration of plasmid pIntTrp1Cup1Gs at the chromosomal
TRP1 locus; this plasmid contains the CUP1
copper-inducible promoter (region
460 to
30) fused to the coding
region of rat Gs
. Plasmid pADHprACVLeu was constructed
by ligating the XbaI-HindIII fragment of dog type
V (12) adenylyl cyclase (in pCDNA1) into the
XbaI-HindIII sites of pVT100U and replacing the
EcoRI-HpaI fragment of pVT100U (containing the
URA3 gene) with the LEU2 gene (HpaI
fragment) from YEP13.
Construction of Plasmid Libraries of Mutant Type V Adenylyl Cyclase-- Randomly mutated libraries of plasmid pADHprACVLeu were generated by passage in the mutator bacterial strain XL1 Red (Stratgene). Libraries with different extents of mutagenesis were prepared by isolating plasmids after 24, 48, and 72 h of growth in this strain. Libraries containing random mutations in the C1 or C2 domains were constructed by error-generated PCR mutagenic techniques. For the C1 domain, sense (nucleotides 1125-1144) and antisense (nucleotides 1807-1826) primers were used and the PCR products were digested with NcoI and NdeI and ligated into these same sites present in the pADHprACVLeu yeast expression plasmid. C2 domain mutant libraries were constructed by replacement of the carboxyl-terminal SphI fragment with the SphI-digested PCR fragment generated with the C2 domain primers (sense = nucleotides 2918-2937, antisense = nucleotides 3754-3773). PCR was performed using standard conditions for Taq polymerase except MgCl2 and deoxynucleotide triphosphate (dNTP) concentrations were as follows (for low, medium, and high level of mutagenesis, respectively): 1.5 mM MgCl2, 0.2 mM dNTP; 7 mM MgCl2, 1 mM dNTP; 7 mM MgCl2, 1 mM dCTP, 1 mM dGTP, 0.2 mM dATP, and 0.2 mM dTTP. PCR was performed for 30 cycles (1 min at 94 °C, 1 min at 45 °C, 1 min at 72 °C).
Selection of Mammalian Type V Adenylyl Cyclase Mutants in
Yeast--
Plasmid libraries were introduced into yeast strain 12229 by standard yeast transformation protocols using lithium acetate. Transformants were plated on yeast plates containing CM/Leu medium supplemented with 100 µM CuSO4, 200 µM forskolin, and 2% dimethyl sulfoxide and incubated at
30 °C. Plasmids were recovered from yeast using Wizard Clean-up
columns (Promega) and introduced into bacteria by electroporation as
per manufacturer's specifications (Bio-Rad). Recovered plasmids were
reintroduced into 12229 and TC41-1 yeast strains by transformation and
replica-plated.
DNA Sequencing-- Sequencing of the adenylyl cyclase mutants was performed using Thermal Sequenase (Amersham Pharmacia Biotech) as per procedures supplied by the manufacturer. In some cases, automated sequencing was performed using an Applied Biosystems sequencer (Applied Biosystems, division of Perkin-Elmer).
Retesting Mutations in Yeast-- Plasmids encoding mutant adenylyl cyclases localized (by sequencing) to the C1 region were produced by ligating the NcoI-NdeI fragment from isolated mutant plasmids into NcoI-NdeI-digested parental plasmid pADHprACVLeu. Plasmids encoding mutant adenylyl cyclases mapping to the C2 region were produced by replacing the SphI-HindIII fragment (encoding the carboxyl terminus of type V adenylyl cyclase) in wild type plasmid pADHprACVLeu with the SphI-HindIII fragment of the isolated mutant plasmid. In all cases, the replacement fragment contained only a single point mutation. These plasmids were reintroduced into 12229 and TC41-1 yeast strains by transformation and replica-plated to verify that the mutation correlated with the growth phenotype of the originally isolated mutant plasmid.
Generation of Recombinant Baculovirus-- Generation of recombinant baculovirus was performed using the fastbac system (Life Technologies, Inc.) as per manufacturer's specifications. For the production of recombinant baculovirus encoding the wild type type V adenylyl cyclase, the BamHI fragment containing the entire coding region of dog type V was ligated to the BamHI-digested pfastbac transfer vector to generate pfastbacACV. Viruses encoding mutant adenylyl cyclases mapping to the C1 region were produced by ligating the NcoI-NdeI fragment from isolated mutant pADHprACVLeu yeast expression plasmids into NcoI-NdeI-digested pfastbacACV. Viruses encoding mutant adenylyl cyclases mapping to the C2 region were produced by replacing the SphI-HindIII fragment (encoding the carboxyl terminus of type V adenylyl cyclase) from pfastbacACV with the SphI-HindIII fragment of the isolated mutant pADHprACVLeu yeast expression plasmids.
Sf9 Cell Culture and Preparation of Cell Membranes-- Procedures for the culture of Sf9 cells and the amplification of recombinant baculovirus have been outlined by Summers and Smith (28). Sf9 membranes containing individual adenylyl cyclase isoforms were prepared as described elsewhere (29).
Purification of G Protein Subunits--
Recombinant
Gs and Gi
were synthesized in bacteria
and purified as described by Lee et al. (30). Protein
concentrations were estimated by staining with Amido Black (31). The
subunits were activated by incubation with 50 mM
Na-HEPES (pH 8.0), 5 mM MgSO4, 1 mM
EDTA, 1 mM dithiothreitol, and 400 µM GTP
S
at 30 °C for 30 (Gs
) or 120 (Gi
) min
(32); unbound GTP
S was removed by gel filtration.
cAMP Assay-- Yeast cells were grown to mid log phase and incubated with appropriate activators/inducers (forskolin/copper) for 3 h, harvested by centrifugation, and cAMP content was measured as described by Uno et al. (33).
Adenylyl Cyclase Assay-- Adenylyl cyclase activity was measured using the procedure described by Smigel (34). All assays were performed for 10 min at 30 °C in a final volume of 100 µl containing 20 µg of membrane protein and a final concentration of 10 mM MgCl2.
Adenylyl Cyclase Purification and Immunoblotting-- Wild type and mutant type V adenylyl cyclase proteins were purified from Sf9 membranes using published methods (9). Samples were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted as described (29), using a primary rabbit antibody specific for type V/VI adenylyl cyclase (ACV/VI) (35) and a secondary 125I-conjugated anti rabbit antibody (NEN Life Science Products). Proteins were visualized on a PhosphorImager (Molecular Dynamics).
Gs Binding Assay--
The labeled
Gs
for binding experiments was prepared by incubating
~50 pmol of Gs
with 1.2 µM
[35S]GTP
S (1250 Ci/mmol) for 1 h at 30 °C in
20 mM Na-HEPES (pH 8.0), 5 mM
MgSO4, 1 mM dithiothreitol, and 1 mg/ml bovine
serum albumin in a final volume of 80 µl. The reaction mixture was
then gel-filtered through a Sephadex G-50 column to remove free
[35S]GTP
S. The Gs
binding assay was
performed by mixing 20 µg of Sf9 cell membranes expressing
wild type or mutant adenylyl cyclase, 80 fmol of
[35S]GTP
S-Gs
(final concentration 4 nM) and unlabeled GTP
S-activated Gs
as
described previously (5, 36).
Data Analysis--
Data were analyzed using the GraphPad Prizm
program. Minimum values for the EC50 for Gs
stimulation were estimated from the dose-response curves; for curves
that did not approach saturation, values represent concentrations of
Gs
that stimulate enzymatic activity half as much as the
highest concentration of Gs
tested (3 µM).
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RESULTS |
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Yeast (Saccharomyces cerevisiae) adenylyl cyclase
(encoded by the CYR1 locus) is an essential gene product;
cyr1 strains lacking a functional cyclase are non-viable but
can be propagated in the presence of cAMP in the growth medium. As
shown in Fig. 1, expression of the
mammalian type V (ACV) adenylyl cyclase (under the control of the yeast
ADH promoter) on a multicopy episomal plasmid can conditionally rescue the lethal phenotype of the
CYR1-deleted strain TC41-1. The expression conditions
employed have allowed us to discern the different regulatory states of
the type V adenylyl cyclase based on growth characteristics of the
yeast. In the absence of cyclase activators, TC41-1 cells expressing
ACV fail to grow, presumably due to low cAMP levels produced by this
isoform under basal conditions (0.02 pmol of cAMP/mg of protein).
However, under activating conditions (in the presence of 100 µM forskolin in the medium or coexpression of
Gs), these cells now grow. Growth was accompanied by an
increase in intracellular cAMP levels (0.25 pmol of cAMP/mg of protein
for either condition). In vitro, ACV is synergistically
activated by Gs
and forskolin in combination, and under
these conditions we were surprised to observe a no growth phenotype
rather than robust growth of this yeast strain despite measuring
enhancement of intracellular cAMP levels (1.2 pmol of cAMP/mg of
protein).
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The observed failure of the yeast strain expressing ACV to grow under
concomitant activation of the cyclase by forskolin and Gs suggested a strategy to isolate adenylyl cyclase
mutants defective in their response to Gs
. We reasoned
that Gs
-insensitive mutants should represent a subset of
those that would allow for growth in the presence of Gs
and forskolin. Toward this end, we have constructed libraries encoding
randomly generated mutant type V adenylyl cyclases in a yeast
expression vector, introduced these libraries into yeast strain 12229 (a derivative of TC41-1 expressing rat Gs
under the
control of the CUP1 copper-inducible promoter), and selected
for transformants that grow in the presence of forskolin and copper.
Several hundred colonies were isolated from >5 × 106
transformants derived from nine libraries; three libraries differing in
the extent of mutagenesis were generated for each target region of ACV
(C1a, C2, and the entire coding region).
Positive colonies selected in these genetic screens were replica-plated
on medium lacking forskolin to assess growth in the presence of
differing levels of expressed Gs (controlled by the
concentration of copper in the growth medium) alone. For mutants that
exhibited reduced sensitivity to Gs
stimulation (observable by lack of growth in the presence of Gs
),
plasmids encoding the mutant cyclases were recovered and introduced
into TC41-1 cells to assess their sensitivity to stimulation by
forskolin and back into the parental yeast strain, 12229, to verify
that the growth phenotype segregated with the mutant adenylyl cyclase plasmid.
Fig. 2 depicts the growth characteristics
of 13 independently isolated mutants defective in responses toward
Gs. All of these mutants (isolated for their ability to
grow in the presence of Gs
and forskolin stimulation)
fail to grow in the presence of Gs
levels sufficient to
obtain growth for the wild type ACV-expressing cells; maximal
activation of the CUP1 promoter by high concentration of
copper in the medium, however, will elevate Gs
levels
enough to permit growth of yeast expressing some of these mutant
cyclases. All but one mutant (D424N) impart a growth phenotype in the
presence of forskolin, consistent with a defect limited to
Gs
regulation. Growth of yeast (expressing these mutants
or wild type) is slower at lower concentrations of forskolin and not
detected at concentrations below 10 µM (data not
shown).
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Twenty-five independent mutants2 were subjected to DNA sequence analysis to localize the sites of mutations responsible for the observed phenotypes. Eleven positions (the C1a and C2 domains of the cyclase containing 2 and 9 sites, respectively) were identified, as shown in Fig. 3. Several point mutants were independently recovered multiple times from different mutant libraries (F379L and F1093S, four times; L967P and G1016S, three times; N1013D and N1094D, two times), and two positions were found mutated to two different amino acids (Leu-967 to Pro or Arg, and Asn-1094 to Asp or Tyr). As depicted in Fig. 3, all mutated positions are highly conserved among all mammalian adenylyl cyclase family members.
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We next examined the biochemical properties of these mutant type V cyclases to determine whether defective regulatory properties of these mutants could account for the altered growth phenotypes observed when they are expressed in yeast. Unfortunately, characterization of the regulatory properties of mutant or wild type adenylyl cyclase in yeast homogenates or membranes was not possible due to extremely low enzymatic activities of these preparations. Therefore, the mutant adenylyl cyclase constructs were introduced into recombinant baculoviruses and overexpressed in Sf9 cells; membranes from these cells were then used as a source of cyclase for biochemical studies. To address potential differences in relative levels of active cyclase protein, the wild type and mutant adenylyl cyclase proteins were purified; equivalent amounts of these cyclases (based on activity measurements) were analyzed by Western blotting with an anti-AC5/6 antibody (data not shown). For comparative purposes, levels of active cyclase protein were normalized with respect to that of the wild type enzyme and are summarized in Table I; the values range from 43% to 385% of wild type and may reflect differences in expression levels or stabilities of the mutant cyclases.
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C1a Mutants--
As shown in Fig.
4, both the D424N and F379L mutants have
reduced responses to stimulation by Gs compared with the
wild type enzyme, correlating well with the growth phenotypes observed in S. cerevisiae. The rightward shift in the dose-response
curves for both of these mutants indicates a reduced affinity for
Gs
relative to the wild type enzyme (EC50
values for wild type, F379L, and D424N are 101, >700, and >600
nM, respectively). We could not determine whether the
maximal stimulation by Gs
was also reduced by these
mutations, because we were unable to reach saturating Gs
concentrations for the mutant type V adenylyl cyclases.
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C2 Mutants--
The majority of the
Gs-insensitive mutants that were isolated in yeast were
localized to the C2 domain of ACV. Biochemical analysis of
these cyclase mutants, after expression in Sf9 cells, reveals
that all are defective in responding to stimulation by Gs
. As shown in Fig. 7,
all mutants have reduced responses to stimulation by Gs
compared with the wild type enzyme. EC50 values of these
mutants for Gs
are shown in Table
II.
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DISCUSSION |
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The 25 Gs-defective adenylyl cyclase mutants
isolated represent point mutations at 11 positions that are highly
conserved among all the identified adenylyl cyclase isoforms; the
mutations are localized to the cytoplasmic regions (C1a and
C2) of the molecule. These mutations can be placed into two
general groups based on their biochemical properties. The D424N, L967P
and L967R are defective in some of their responses to Gs
but also display other regulatory abnormalities. The defects of the
second group of mutants are limited to Gs
responses (in
all aspects tested), including Gs
binding, activation,
and synergistic interaction with forskolin activation (and, for those
mutants examined, stimulation of forskolin binding); the biochemical
properties of these mutants are consistent with the involvement of
these residues in the Gs
binding site.
Based on the homologies among the cytoplasmic domains of mammalian
adenylyl cyclase isoforms and between the C1 and
C2 regions within a given cyclase isoform, all but the
Leu-967 mutants identified in this study can be mapped to positions on
the recently solved crystal structure of the ACII C2
homodimer (22). Asp-424 is analogous to the conserved Asp-923 of the B
chain of the ACII homodimer located on helix 2. Closer inspection of
this aspartate residue reveals that its carboxyl side chain intimately
associates with Asn-1124 and Arg-1125 of the C2 domain
(ACII residues 1012 and 1013 of the A chain). Mutation of this
aspartate would disrupt its ability to hydrogen-bond to the backbone
nitrogen of these residues, thereby decreasing the interaction of the
two cytoplasmic domains. In light of the finding that both
Gs and forskolin activate adenylyl cyclase by increasing
the affinity of C1 for C2 (37, 38), the
predicted consequence of mutating Asp-424 would be precisely what was
observed for the D424N mutant: reduced sensitivity to stimulation by
either forskolin or Gs
alone, and a retention of the
ability to synergistically integrate these stimulatory inputs.
The second group of mutants identified serves to identify three regions
of the adenylyl cyclase molecule critical for its interaction with
Gs. Phe-1093 and Asn-1094 reside at the junction between
helix 3 and
strand 4; Phe-1006, Asn-1013, Gly-1016, Val-1017,
Arg-1021, and Val-1022 lie at the base of (and loop preceding)
helix 2. The residues in these two regions are highlighted (Fig.
9). The third region is in
C1a (Phe-379) and is represented in dark green
in Fig. 9 (the actual residue highlighted is the amino-terminal most
residue of the C2 homodimeric structure, Glu-875, which is
2 residues carboxyl-terminal to the homologous residue of Phe-379). All
of these residue (except Gly-1016) are located on the same surface of
the adenylyl cyclase protein displaying a localized binding site for
Gs
docking. Residue Gly-1016 is buried, and it is likely
that G1016S was isolated as a Gs
-insensitive mutant in
our genetic selection because this substitution would undoubtedly
disrupt the geometry of the exposed surface of
helix 2.
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All mutations isolated in this study are located in positions highly
homologous among all Gs-regulated adenylyl cyclases. It
is interesting to note that the one exception, residue Asn-1013, is
absolutely conserved among types I-VIII, but differs in type IX (Tyr)
and Drosophila rutabaga (Ser) cyclases. This may explain the
relatively less drastic biochemical defect observed for the isolated
N1013D mutant; it retained much of its Gs
binding and
activation (in the presence of forskolin) despite showing decreased
binding to and stimulation by Gs
alone.
The fact that the Gs binding site that we have
determined by the mutational analysis comprises residues located in
both C1 and C2 domains is consistent with the
observation that Gs
increases the affinity of the
cytoplasmic domains for each other (37, 38). That most of the
Gs
-insensitive mutants isolated in this study map to
residues in the C2 domain is consistent with the
observation that recombinant soluble C2 domain constructs can bind to Gs
in vitro, whereas the
C1 domain cannot but can be covalently attached to
Gs
in the presence of chemical cross-linkers (39). A
recent study utilizing alanine substitutions of cyclase residues has
defined a number of mutations that disrupt Gs
activation
of soluble C1 (type I) and C2 (type II) cyclase constructs analogous to several isolated in our random mutagenic approach (40). Our findings confirm the importance of C1
residue Phe-379 (ACV numbering), and C2 residues (Arg-1021,
Val-1022, Phe-1093, and Asn-1094) in Gs
activation, and
further demonstrate the importance of these residues for
Gs
binding and their function in the context of the
full-length membrane-bound enzyme.
It is unclear how Gs precisely binds to the cyclase
because the three-dimensional structure of Gs
is not yet
solved; however, molecular modeling using the available
crystallographic structure of Gi
(41) suggests a
possible orientation of this interaction. In the Gi
1, a
hydrophobic pocket is formed by Ile-212, Phe-215, and Trp-258 when GTP
is bound. The homologous amino acids in Gs
could
accommodate Phe-1093 in type V adenylyl cyclase, thereby orienting
residues (shown to be important for the ability of Gs
to
activate adenylyl cyclase; see Refs. 23 and 24) toward
helix 2 and
Phe-379 of the cyclase. We are currently using our yeast genetic system
to identify second-site suppressors, i.e. mutations in
Gs
that have gained the ability to stimulate the
Gs
-insensitive adenylyl cyclase mutants, in an effort to
identify the precise interactions between Gs
and its
binding site on adenylyl cyclase.
Expression of mammalian type V adenylyl cyclase in a cyclase-deficient
mutant of S. cerevisiae has allowed us to take a genetic approach toward elucidating the structural basis underlying the regulation of adenylyl cyclase isoforms. The utility of this genetic system is evidenced by the isolation of mutant adenylyl cyclases defective in their regulation by Gs. The genetic system
should be readily applicable to the selection of additional adenylyl cyclase mutants defective in their responses to other regulators such
as Gi
, G protein
subunits, or protein kinases,
thus leading to the identification of the binding sites for these
molecules.
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ACKNOWLEDGEMENTS |
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We thank V. Siguel for excellent technical assistance, Dr. R. Green for the rabbit antibody specific for type V/VI adenylyl cyclase, Dr. J. Stuckey for help with molecular modeling analysis of the adenylyl cyclase structure and figures, and Dr. R. Neubig for help with data analysis and thoughtful discussion.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM53645, Michigan Gastrointestinal Hormone Research Core Center Grant DK34933, Michigan Diabetes Research and Training Center Grant DK20572, and the Burroughs Wellcome Foundation.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: 3301E MSRB III, Dept.
of Biological Chemistry, University of Michigan Medical School, Ann
Arbor, MI 48109-0636. Tel.: 734-647-3634; Fax: 734-763-7799.
1
The abbreviations used are: G protein,
heterotrimeric guanine nucleotide-binding regulatory protein;
Gs, the
subunit of the G protein that stimulates
adenylyl cyclase; Gi
, the
subunit of the G protein
that inhibits adenylyl cyclase; GTP
S, guanosine
5'-O-(2-thiodiphosphate); PCR, polymerase chain reaction; AC, adenylyl cyclase.
2 Mutant F379L was independently isolated from three libraries, two times from the PCR-targeted C1 domain library, and once each from the 24-h and 48-h randomly mutated libraries. Mutant D424N was isolated from the 72-h randomly mutated library. Mutant L967P was independently isolated from both the 24-h randomly mutated library and the PCR-targeted C2 domain library. All other mutants were isolated from the PCR-targeted C2 domain library.
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REFERENCES |
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