(Received for publication, April 15, 1997, and in revised form, June 20, 1997)
From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041
Forskolin- and Gs-stimulated
adenylyl cyclase activity is observed after mixture of two
independently-synthesized ~25-kDa cytosolic fragments derived from
mammalian adenylyl cyclases (native Mr ~ 120,000). The C1a domain from type V adenylyl cyclase
(VC1) and the C2 domain from type II adenylyl
cyclase (IIC2) can both be expressed in large quantities
and purified to homogeneity. When mixed, their maximally stimulated
specific activity, 150 µmol/min/mg protein, substantially exceeds
values observed previously with the intact enzyme. A soluble,
high-affinity complex containing one molecule each of VC1,
IIC2, and guanosine 5
-O-(3-thiotriphosphate) (GTP
S)-Gs
is responsible for the observed enzymatic
activity and can be isolated. In addition, GTP
S-Gs
interacts with homodimers of IIC2 to form a heterodimeric
complex (one molecule each of Gs
and IIC2)
but not detectably with homodimers of VC1. Nevertheless,
Gs
can be cross-linked to VC1 in the
activated heterotrimeric complex of VC1, IIC2,
and Gs
, indicating its proximity to both components of
the enzyme that are required for efficient catalysis. These results and
those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate
formation of a single, high-activity catalytic site at the interface
between C1 and C2.
Eleven distinct isoforms of mammalian adenylyl cyclase have been
identified to date, and the regulatory properties of several of these
proteins have been characterized extensively (1, 2). Although there is
remarkable variation in the responsiveness of these enzymes to
inhibitory effects of the
Gi1 proteins
and to such agents as G protein
subunits and Ca2+,
the catalytic activity of all of the known isoforms is stimulated by
the
subunit of Gs and, presumably nonphysiologically,
by the diterpene forskolin. The adenylyl cyclases share a unique structure for an enzyme, resembling transporters such as the
P-glycoproteins topographically. They are intrinsic membrane proteins
by virtue of their two large hydrophobic domains, each of which is
hypothesized to contain six membrane-spanning helices. The first of
these hydrophobic regions follows a short amino-terminal sequence and
precedes a roughly 40-kDa cytoplasmic domain (C1). The
second hydrophobic region separates C1 from a second
cytosolic domain (C2) of comparable size. Each of the two
cytosolic domains includes a sequence of 200-250 amino acid residues
that is typically 50% similar to its consort, 50-90% similar to the
corresponding domains of other adenylyl cyclase isoforms, and 20-25%
similar to the catalytic domains of membrane-bound and cytosolic
guanylyl cyclases.
Detailed biochemical characterization of adenylyl cyclase is impaired
by the insolubility, instability, and sparsity of the native enzyme, as
well as our incapacity to express necessary amounts of the protein in
heterologous systems. To overcome these hurdles we have synthesized (in
Escherichia coli) portions of the two cytosolic domains of
adenylyl cyclase in the absence of the remainder of the protein, first
as a 55-kDa chimeric fusion protein containing the C1a
domain of type I adenylyl cyclase linked to the C2 domain
of the type II enzyme (3, 4). The specific activity of this engineered
enzyme is remarkably high, it is soluble in the absence of detergent,
and it is adequately stable. Importantly, it is activated
synergistically by Gs and forskolin and inhibited by
so-called P-site inhibitors and the G protein
subunit complex, providing ample justification to pursue investigation of this and
similar artificial entities. To overcome the remaining hurdle of a
relatively low level of accumulation of the chimera in bacterial cytosol, we and others prepared the two cytosolic domains of adenylyl cyclase as distinct entities and found that enzymatic activity with
similar regulatory properties can be reconstituted by simple mixture of
the two roughly 25-kDa proteins (5, 6). Large amounts (50-100 mg or
more) of the C2 domain of type II adenylyl cyclase can be
prepared readily, but similar results are difficult to achieve with the
C1a domain of the type I enzyme. We have now extended this
approach by utilizing a fragment of the C1 domain of type V
adenylyl cyclase, which can be expressed in reasonable (but not
exuberant) quantities, and we have characterized the interactions of
the two cyclase fragments with each other and with
Gs
.
DNA containing nucleotides 961-2010
(amino acid residues 321-670) of canine type V adenylyl cyclase (7)
was generated with the polymerase chain reaction using oligonucleotides
A: 5-CCATGGCTGAGGTCTCCCAG-3
; and B:
5
-TTGCGGCCGCGGATCCGGTCAGGCTCCCTGAAGG-3
, as primers. Note that
oligonucleotide A is 5
to a unique NcoI site in the
cDNA for canine type V adenylyl cyclase and includes the codon for Met364. Further truncations of this fragment were generated
by taking advantage of unique restriction sites for AvrII
(at nucleotide 1815), BstBI (at nucleotide 1858), and
Bsu36AI (at nucleotide 1900) to create
VC1(364-606), VC1(364-620), and
VC1(364-635), respectively. VC1(364-567),
VC1(364-591), and VC1(364-591)Flag were
generated with the polymerase chain reaction using the following pairs
of oligonucleotide primers: A and 5
-TTCTCCGGATCCAAGCTTGCAGCGCAGGA-3
, A and 5
-GATTCGAAGCTTGTGCCCAATGGAG-3
, and A and
5
-GCTAATTAAGCTTGTCATCGTCGTCCTTGTAGTCGTGCCCAATGGAGTTG-3
. The last
construct contains a carboxyl-terminal Flag epitope (Kodak), DYKDDDDK,
as well as a cleavage site for enterokinase. All of the VC1
constructs were ligated into pQE60-H6 (8) with NcoI and
HindIII. Thus, each of the encoded proteins contains a
hexa-histidine tag at its amino terminus, followed by the residue
corresponding to Met364 of canine type V adenylyl cyclase,
and terminates at the stop codon contained in pQE60-H6. We will
subsequently designate these proteins by reference to their
carboxyl-terminal residue.
E. coli
strain BL21 was co-transformed with pREP4 and pQE60-H6-VC1
(various) and grown overnight at 30 °C in 200 ml of LB medium
containing ampicillin (50 µg/ml) and kanamycin (50 µg/ml). This
culture was used to inoculate 10 liters of T7 medium (8), which was
incubated at 30 °C until OD600 reached 1.3. Synthesis of
VC1 was then induced with 30 µM isopropyl
thiogalactoside, and incubation was continued for 4 h at room
temperature. Cells were harvested by centrifugation, frozen in liquid
N2, and stored at 70 °C. Frozen cells were pulverized
and suspended in 750 ml of buffer A (50 mM Tris-HCl,
pH 8, 120 mM NaCl, 1 mM
-mercaptoethanol, and a mixture of protease inhibitors (4)) prior to incubation with 0.25 µg/ml lysozyme in buffer A for 30 min at 4 °C and subsequently 8 µg/ml DNase and 1 mM MgCl2 in buffer A for an
additional 30 min. Cellular debris was removed by centrifugation at
100,000 × g for 40 min at 4 °C.
All purification steps were performed at 4 °C. The supernatant (750 ml) was applied to a 5-ml metal chelate column (Talon, CLONTECH) equilibrated in buffer A, and the column
was washed with 20 volumes of buffer A containing 500 mM
NaCl and 10 volumes of buffer B (20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM -mercaptoethanol, and protease
inhibitors). H6-VC1 was eluted from the column in 2-ml
fractions with buffer B containing 100 mM imidazole. Peak fractions were pooled, diluted with 2 volumes of buffer C (20 mM NaHepes, pH 8.0, 20 mM NaCl, 2 mM dithiothreitol, and protease inhibitors) and applied to
a Mono Q 5/5 FPLC column (Pharmacia). This column was washed with 5 ml
of buffer C and protein was eluted with a 25-ml linear gradient of NaCl
(20-300 mM in buffer C). Peak fractions (about 150 mM NaCl) were pooled and concentrated to 3 mg/ml unless
noted otherwise.
The C2 domain of type II adenylyl cyclase
(IIC2) was expressed in E. coli and purified as
described previously (5), as was bovine Gs (short form)
(8). Gs
was activated with GTP
S by incubation with 50 mM NaHepes, pH 8.0, 10 mM MgSO4, 1 mM EDTA, 2 mM dithiothreitol, and 800 µM GTP
S for 30 min at 30 °C.
Proteins (Gs and/or the
adenylyl cyclase domains) were applied (200-µl sample volumes) to a
Superdex 200 column (HR10/30; Pharmacia) or to Superdex 75 (HR10/30)
and 200 columns in tandem. Samples were eluted with 20 mM
NaHepes, pH 8.0, 2 mM MgCl2, 1 mM
EDTA, 2 mM dithiothreitol, 100 mM NaCl, and
(where indicated) 50 µM forskolin. The flow rate was 0.3 ml/min and fractions were 400 µl unless otherwise indicated.
Sedimentation equilibrium
analysis was performed by centrifugation of samples (100 µl) for
60 h at 4 °C in a Beckman TL100 centrifuge as described
previously (9). The buffer contained 50 mM NaHepes, pH 8.0, 10 mM MgCl2, 1 mM EDTA, 20 mM -mercaptoethanol, 50 mM NaCl, 50 mM 6-O-(3
-(piperidino)propionyl)forskolin,
and 5 mg/ml dextran T40 to provide density stabilization. Immediately after centrifugation, 7-µl fractions were collected with a Brandel microfractionator. The concentration of
[35S]GTP
S-Gs
in each fraction was
determined by liquid scintillation spectrometry. The concentration of
adenylyl cyclase in each aliquot was determined by quantification of
catalytic activity under linear conditions with addition of either 1 µM VC1(591) or IIC2 in the presence of 50 µM forskolin and 0.2 µM
GTP
S-Gs
. The addition of exogenous
GTP
S-Gs
and either the C1 or
C2 domain of adenylyl cyclase was necessary to assure
linearity of activity throughout the range of concentrations found in
the gradient. Molecular weights were calculated from the slope of a
plot of ln C/Co versus r2, where C is the protein concentration in
a given fraction, Co is the initial concentration,
and r is the radial distance in the centrifuge (cm). Similar
results were obtained for all conditions at two different rotational
velocities.
Cross-linking studies were performed
utilizing the bifunctional amine-coupling reagent disuccinimidyl
suberate (Pierce). Purified proteins were diluted into a buffer
containing 20 mM NaHepes (pH 8.0), 1 mM EDTA,
and 2 mM MgCl2. Cross-linking was initiated by addition of freshly prepared disuccinimidyl suberate (100 µM final concentration) and allowed to progress for 15 min at room temperature. Reactions were terminated with SDS-PAGE sample
buffer containing 10 mM glycine and 20 mM
dithiothreitol, and proteins were resolved by SDS-PAGE. Proteins were
visualized by immunoblotting using antibodies specific for
H6-VC1(364-591)Flag (anti-Flag), IIC2-H6 (10),
or Gs (11).
Adenylyl cyclase activity was measured as described by Smigel (12). All assays were performed for 10 min at 30 °C in a volume of 100 µl. In reconstitutive assays the final concentration of IIC2 was at least 1-5 µM to maintain linearity with variable concentrations of VC1. The final concentration of ATP was 1 mM unless stated otherwise, and an ATP regenerating system was used only when crude preparations of enzyme were assayed.
The C1 domain of type V adenylyl cyclase was truncated at its carboxyl terminus in attempts to obtain a pure, stable protein with high specific enzymatic activity when reconstituted with IIC2. Bacterial lysates containing constructs as large as VC1(670), for example, supported high forskolin-stimulated adenylyl cyclase activity (>50 nmol/min/mg in the lysate), but immunoblot analysis of SDS-PAGE gels indicated contamination with several proteolytic products (data not shown). Extracts containing truncations VC1(635), VC1(621), or VC1(606) displayed progressively less such contamination, but susceptibility to degradation was again high with VC1(567); the latter protein is analogous to the C1 domain of type I adenylyl cyclase described previously (5). However, extracts of bacteria expressing VC1(591) or its carboxyl-terminally Flag-tagged counterpart, VC1(591)Flag, contained only a single significant proteolytic product (molecular mass ~ 25 kDa; mass of VC1(591) ~ 28 kDa), and its accumulation was minimized by shortening the length of time for expression to 4 h. The specific activities of such lysates for reconstitution of adenylyl cyclase activity with IIC2 were typically 50-100 nmol/min/mg extract protein. These values are roughly 1% of those observed with purified native adenylyl cyclases (12-14).
Metal chelate column chromatography (Talon resin) of extracts containing VC1(591)Flag resulted in 100-fold or more purification of the protein with loss of 60% of the total activity (Table I). The remaining activity was not found in the flow-through or column washes. Subsequent Mono Q column chromatography resulted in 2-3-fold purification and removal of the remaining major contaminants. This final product appeared essentially homogeneous when analyzed electrophoretically (Fig. 1) and had a reconstitutive specific activity of 30 µmol/min/mg when assayed with maximally effective concentrations of purified IIC2 and 50 µM forskolin as the sole activator. This purified protein will subsequently be designated VC1.
|
Adenylyl Cyclase Activity Reconstituted from VC1 and IIC2
The adenylyl cyclase activity observed in
mixtures of VC1 and IIC2 resembles that seen
previously with mixtures of IC1 and IIC2 (5,
6); many of the regulatory features that characterize native
membrane-bound adenylyl cyclases are retained. Activities shown in Fig.
2, A, B, and D,
were measured with low concentrations of IIC2 and either
near saturating (2 µM, Fig. 2, A and
B) or variable (Fig. 2D) concentrations of
VC1. Activities shown in Fig. 2C were measured
with low concentrations of VC1 and near saturating (2 µM) concentrations of IIC2. The reconstituted
enzyme is activated by either forskolin (EC50 > 10 µM) or Gs (EC50 ~ 400 nM) (Fig. 2, A and B). Unlike the
mixture of IC1 and IIC2, GTP
S-Gs
could not activate a mixture of
VC1 and IIC2 maximally. The effects of
forskolin and GTP
S-Gs
are synergistic; the presence
of one activator increasing the apparent affinity (EC50) of
the enzyme for the other by 40-fold or more. In the absence of an
activator, the rate of cyclic AMP synthesis is low and the two domains
of the enzyme have an apparent affinity for each other of greater than
5 µM (Fig. 2D, inset). This value is lowered
in the presence of activators; the apparent affinity of VC1
and IIC2 is 0.66 µM with
GTP
S-Gs
, 1.2 µM with forskolin, and 150 nM when both activators are present.
Activation of adenylyl cyclase activity by Gs is
dependent on the nature of the bound nucleotide, but not to the extent usually assumed. GDP-Gs
is only 10-fold less potent than GTP
S-Gs
and is equally efficacious (in the presence
or absence of forskolin (Fig. 2B)). Identical results were
obtained with limiting concentrations of either VC1 or
IIC2 (data not shown).
We have examined the interactions of
VC1 and IIC2 with each other and with
Gs by gel filtration, sedimentation equilibrium centrifugation, and (see below) chemical cross-linking. Gel filtration of VC1 or IIC2 on tandem columns of Superdex 75 and 200 suggests that each of these proteins exists in solution as
roughly 50-kDa homodimers (Fig. 3,
A and B); each individual protein has a molecular mass of approximately 28,000. Similarly, sedimentation equilibrium analysis of IIC2 revealed a molecular weight of 46,000 (Fig. 6D). Gel filtration of a mixture of VC1
and IIC2 in the presence (Fig. 3B) or absence
(not shown) of forskolin revealed a single peak of protein with an
apparent molecular weight of 50,000, while sedimentation equilibrium
analysis of the mixture indicates a molecular weight of 52,000 (Fig.
6B). Because of the similarity of the molecular masses of
VC1 and IIC2, we do not know if these values
represent homodimers, heterodimers, or a mixture of the two.
Mixture of VC1, IIC2, and
GTPS-Gs
(in the presence of forskolin) results in
formation of a high-affinity complex. This is detected by gel
filtration with the appearance of a new peak of optical density,
migrating with an apparent molecular weight of roughly 100,000 (Fig.
3B). SDS-PAGE analysis of fractions obtained by gel
filtration of such a mixture containing an excess of IIC2 indicates that this high molecular weight peak contains all three proteins, with an apparent stoichiometry of 1:1:1 (by scanning densitometry) (Fig. 4). (Note that
essentially all of the VC1 is found in this high molecular
weight peak, presumably indicating that most of the protein is active.)
Similarly, equilibrium sedimentation of such mixtures reveals a
104-107 kDa complex when either Gs
or adenylyl cyclase
activity is monitored (Fig. 6, A and B). The anticipated molecular weight of a 1:1:1 complex of Gs
,
VC1, and IIC2 is 102,000.
Interaction between Gs and IIC2 alone can
also be detected by gel filtration and equilibrium sedimentation.
Incubation of 50 µM
[35S]GTP
S-Gs
with 5, 35, or 250 µM IIC2 causes elution of radioactivity at a
position consistent with progressively higher molecular weights (Fig.
5C). At the highest
concentration of IIC2, the complex is consistent with
molecular mass ~70 kDa. No such interactions were detected between
VC1 and GTP
S-Gs
(Fig. 5A) or
IIC2 and GDP-Gs
(Fig. 5B). With
sedimentation equilibrium, the apparent molecular weight of
Gs
was increased by 24,000 following mixture with IIC2 (but not with VC1) (Fig.
6C) and analysis of adenylyl
cyclase activity revealed a complex between GTP
S-Gs
and IIC2 with mass = 64 kDa. (Note: observation of
enzymatic activity in this experiment required addition of
VC1; the complex of GTP
S-Gs
with
IIC2 does not have detectable adenylyl cyclase activity.) All of these observations indicate formation of a relatively low affinity 1:1 complex between IIC2 and
GTP
S-Gs
, and, thus, that interaction of the adenylyl
cyclase domains with the activated G protein
subunit disrupts the
homodimeric interactions characteristic of VC1 and
IIC2.
Chemical Cross-linking
Although direct interactions between
VC1 and Gs were not detected by gel filtration or
sedimentation equilibrium, chemical cross-linking studies do suggest
that the molecules are at most 11 Å away from each other when tightly
complexed with IIC2. Fig. 7A illustrates the forskolin
(and IIC2-, not shown)-dependent appearance of
a 70-kDa species representing covalent coupling of Gs
and VC1 by the 11-Å cross-linker, disuccinimidyl suberate. Analysis of the same fractions with an anti-Gs
antibody
also reveals a 70-kDa species that appears in a
forskolin-dependent manner. It is unclear in this panel if
this species represents Gs
-VC1,
Gs
-IIC2, or both, since the cyclase domains have similar molecular weights. This was also complicated by the inability of available IIC2 antibodies to detect
IIC2-Gs
complexes by immunoblotting.
Cross-linking studies performed at higher protein concentrations (2 µM VC1, IIC2, and
Gs
) and analyzed with an anti-Gs
antibody
reveal a IIC2-Gs
cross-linked species in the
absence of VC1 (Fig. 7E, lane 4). Moreover, a
large species (Mr ~ 95,000) appears in the
presence of all three proteins and 100 µM forskolin; we
presume this to be the
VC1-IIC2-Gs
heterotrimer (Fig.
7E, lane 7). Observation of both the cross-linked heterotrimer and the IIC2-Gs
heterodimer is
dependent on the presence of GTP
S-activated Gs
(Fig.
7E, lane 3 versus 4 and lane 6 versus 7).
Chemical cross-linking also revealed forskolin-dependent
formation of heterodimers between VC1 and IIC2
(Fig. 7, C and D). Although each domain exists as
a homodimer under nonactivating conditions, formation of heterodimers
occurs in a forskolin (and Gs-)-dependent
manner.
We have expressed and purified a fragment of the C1
domain of type V adenylyl cyclase, consisting of amino acid residues
364-591 and including hexa-histidine and Flag tags at the amino and
carboxyl termini, respectively. Although this protein itself has no
adenylyl cyclase activity, catalysis of cyclic AMP synthesis is
restored by simple mixture of VC1 with an appropriate
fragment from the second cytosolic domain of the enzyme, such as
IIC2. This interaction is similar to that described
previously between IC1 and IIC2 (5, 6); the
major advantages are the yield of VC1, which exceeds that
of IC1 by 20-fold, and the apparent homogeneity of the
product. The adenylyl cyclase activity of the
VC1-IIC2 mixture is stimulated markedly by
either Gs or forskolin, and these two regulators activate the enzyme synergistically when present simultaneously. These
are characteristics of native type II and type V adenylyl cyclases.
A notable difference between this reconstituted adenylyl cyclase and
the native enzymes is the maximal stimulated activity, which typically
exceeds 100 µmol/min/mg in the case of the mixture of VC1
and IIC2; values of 10 µmol/min/mg typify purified
preparations of native enzymes (12-14). Although the source of this
discrepancy is not known, we suspect that the values observed with the
VC1/IIC2 mixture may indeed approximate a true
Vmax for mammalian adenylyl cyclase. Several
factors may cause underestimation of maximal activity when dealing with
a native adenylyl cyclase. Overexpression of these enzymes in Sf9 cells
is plagued by production of nonfunctional protein; detergents are
necessary to maintain solubility of the native proteins but may alter
estimates of specific activity; lengthy purification schemes may cause
denaturation of these labile entities. Alternatively, it is possible
that inhibitory domains have been removed from the soluble constructs
described above or that the membrane spanning domains of the enzymes do
not permit optimal orientation of the interacting cytosolic segments.
Another notable difference between native adenylyl cyclases and the
engineered soluble enzymes (whether or not the cytosolic domains are
linked covalently) is the relatively low (reduced 20-50-fold) apparent affinities of the soluble enzymes for Gs. It is possible that the transmembrane spans, the loops that connect them, and/or residues immediately surrounding the remnants of the C1 and
C2 domains in the constructs utilized may contribute to the
binding site for Gs
. Nevertheless, the essential
features of activation of adenylyl cyclase by the G protein
subunit
are retained.
We demonstrate herein that the C1 and C2
domains of adenylyl cyclase interact to form a catalytically active
adenylyl cyclase and that the apparent affinity of C1 for
C2 is enhanced in the presence of Gs and/or
forskolin. Gel filtration, equilibrium sedimentation, and cross-linking
analyses all demonstrate that Gs
interacts with the
C2 domain of adenylyl cyclase in a GTP-enhanced manner and
that this interaction is further stabilized by C1. Direct
interactions between VC1 and Gs
were not
detected by gel filtration or sedimentation equilibrium. However, these two proteins could be cross-linked by disuccinimidyl suberate in a
IIC2-dependent manner. We further demonstrate
that Gs
, C1, and C2 form a
relatively high-affinity complex with a 1:1:1 stoichiometry. Although
this is not surprising, the similarities in the primary sequence of the
C1 and C2 domains of adenylyl cyclase raised
the possibility of binding sites for two molecules of
Gs
. Several adenylyl cyclase isoforms are inhibited by
Gi
. Although high concentrations of Gi
can apparently compete for the Gs
-binding site,
inhibition of adenylyl cyclase activity by Gi
is not
dependent on Gs
and is not competitive with the
stimulatory
subunit (15). We presume that there is a distinct
binding site for Gi
on certain adenylyl cyclases; Gi
may well be found to interact predominantly with
C1.
Homodimerization of C1 and C2 is difficult to interpret. The phenomenon may clearly be an artifact of protein engineering and have no significance with regard to the membrane-bound native enzyme. However, we have previously detected oligomers of near native adenylyl cyclases in detergent solution, suggesting the possibility of relevance of homodimerization of the cytosolic domains (16).
The relative capacities of GTP- and GDP-bound G proteins
to interact with their effectors has been long debated and may be
dependent on the system in question. However, no such interaction has
probably been examined previously in a system containing such highly
purified proteins. The Gs
, VC1, and
IIC2 proteins utilized herein are all expressed abundantly
in bacteria, greatly facilitating their purification to a high degree.
Significant levels of contaminating nucleotide kinases are extremely
unlikely, obviating such concerns as conversion of GDP to GTP in the
presence of ATP. Nevertheless, the apparent affinity of
GDP-Gs
for adenylyl cyclase is only about 10-fold less
than that of GTP
S-Gs
. The GTPase activity of a G
protein
subunit thus facilitates deactivation of the system by
reducing the affinity of G
for its effector. However,
the affinity of the G protein
subunit complex for
G
is more highly dependent on the nature of the bound
nucleotide. GTP hydrolysis thus greatly favors association of
with
, and it is this interaction that prevents access of either
or
to effectors.
More critical and microscopic analyses of the interactions of the two
cytosolic domains of adenylyl cyclase with each other and with their
regulators are necessary. Although this need has been recognized for a
long time, progress has been greatly limited by the availability and
purity of reagents. Milligram quantities of G protein-regulated
adenylyl cyclase domains may now be prepared readily, as can a
high-affinity complex of Gs with the crucial components
of the cyclase. We hope that all relevant tools can now be utilized to
understand the complex regulatory features of these interesting
enzymes.
We thank Julie Collins and Jeff Laidlaw for
superb technical assistance and Alex Duncan for generously providing
purified wild type Gs.