Compartmentation of Cyclic Adenosine 3',5'-Monophosphate Signaling in Caveolae
Carsten Schwencke,
Manabu Yamamoto,
Satoshi Okumura,
Yoshiyuki Toya,
Song-Jung Kim and
Yoshihiro Ishikawa
Cardiovascular & Pulmonary Research Institute Allegheny
University of the Health Sciences Pittsburgh, Pennsylvania
15212
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ABSTRACT
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The cAMP-signaling pathway is composed of multiple
components ranging from receptors, G proteins, and adenylyl cyclase to
protein kinase A. A common view of the molecular interaction between
them is that these molecules are disseminated on the plasma lipid
membrane and random collide with each other to transmit signals. A
limitation to this idea, however, is that a signaling cascade involving
multiple components may not occur rapidly. Caveolae and their principal
component, caveolin, have been implicated in transmembrane signaling,
particularly in G protein-coupled signaling. We examined whether
caveolin interacts with adenylyl cyclase, the membrane-bound enzyme
that catalyzes the conversion of ATP to cAMP. When overexpressed in
insect cells, types III, IV, and V adenylyl cyclase were localized in
caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated
with adenylyl cyclase in tissue homogenates and copurified with a
polyhistidine-tagged form of adenylyl cyclase by Ni-nitrilotriacetic
acid resin chromatography in insect cells, suggesting the
colocalization of adenylyl cyclase and caveolin in the same
microdomain. Further, the regulatory subunit of protein kinase A
(RII
, but not RI
) was also enriched in the same fraction as
caveolin. Gs
was found in both caveolin-enriched and
non-caveolin-enriched membrane fractions. Our data suggest that the
cAMP-signaling cascade occurs within a restricted microdomain of the
plasma membrane in a highly organized manner.
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INTRODUCTION
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The rapid amplification of cAMP signaling upon agonist binding is
well known (1). This involves the sequential activation of the
receptor, G protein, and adenylyl cyclase at the plasma membrane, and
the production of a second messenger cAMP. cAMP, a diffusible second
messenger, leads to activation of the cAMP-dependent protein kinase
[protein kinase A (PKA)], a heterotetramer composed of two regulatory
subunits (R) and two catalytic subunits (C) (2). PKA initiates a
cascade of multiple protein phosphorylation events, leading to, for
example, enhanced contractility in the heart.
A prevailing molecular view of the receptor/G-protein/effector
interaction in cells is based upon the random collision theory, in
which proteins are floating randomly on the lipid membrane surface and
contact with the target molecule with the specificity at the sites of
protein-protein interaction (3, 4). A major limitation to this theory,
however, is that a rapid signaling cascade involving multiple proteins
may not be efficiently processed, particularly when these signaling
molecules are scarce. Indeed, adenylyl cyclase is a rare component of
the cell membrane, constituting only approximately 0.001% of the total
membrane protein. Accordingly, a fundamental question in signal
transduction is how particular signaling molecules are rapidly and
specifically linked to each other within the plasma membrane.
Compartmentation of molecules by a scaffolding protein(s), which places
multiple related signaling molecules to specific intracellular sites,
has been demonstrated in some cases of signaling (for review see Ref.
5); mechanisms for recognizing phosphotyrosine (SH2/SH3 domains) and
various peptide motifs [PDZ, WW, and pleckstrin homology (PH)
domains] have been well recognized. Although the exact mechanisms
and/or scaffolding proteins are not known for cAMP signaling, several
observations have suggested that some components within the cAMP-
signaling pathway are colocalized to discrete regions of the plasma
membrane (for review see Refs. 6, 7). These suggestions are based on
early studies demonstrating that locally applied catecholamines can
achieve maximum contraction of cardiac papillary muscle without changes
in whole-tissue cAMP concentrations (8) or reconstitution studies from
Rosss laboratory (9). Recent observations using the whole-cell
patch-clamp method demonstrated that catecholamine receptors are
functionally coupled to nearby Ca2+ channels via local
elevations of cAMP concentrations in cardiomyocytes (10). Some adenylyl
cyclase isoforms are prominently stimulated by capacitative
Ca2+ entry, whereas ionophore-mediated Ca2+
release has no significant effect on their activity (11). These
observations suggest that the components within the cAMP-signaling
pathway, including adenylyl cyclase, are colocalized within the cell
membrane, thereby allowing rapid and preferential modulation of cAMP
production within a defined microenvironment.
Caveolae, flask-shaped plasmalemmal vesicles, represent a
subcompartment of the plasma membrane that exists in most cell types
including endothelial cells, fibroblasts, adipocytes, and myocytes
(12, 13, 14). Caveolin is a major protein component of caveolae. Multiple
members of the caveolin gene family have been identified (caveolin-1
and -ß, caveolin-2 and -3) that differ in tissue distribution (14).
The recent identification of various G protein
- and ß
-subunits
and their functional interaction with caveolin suggest that caveolae
may participate in transmembrane signaling, in particular, G
protein-coupled receptor signaling (15, 16, 17). We thus investigated the
possible interaction of adenylyl cyclase and other molecules within the
cAMP-signaling pathway with caveolin. We used multiple adenylyl cyclase
isoforms that have distinct tissue distribution and biochemical
properties but are all stimulated by Gs
(reviewed in Refs. 1, 18).
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RESULTS AND DISCUSSION
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Isolation of Endogenous and Recombinant Caveolin-Enriched Membrane
Fractions
Caveolin-enriched membrane fractions were obtained from cultured
COS-7 cells using the sucrose density gradient centrifugation (17, 19).
Ponceau S staining revealed that total cellular protein remained mainly
in the bottom-loaded sucrose layer (Fig. 1A
). These fractions retained
approximately 97% of total cellular protein; this value was similar in
our subsequent purification experiments, regardless of the starting
material (mammalian cells, insect cells, or tissues). As shown by the
pattern of distribution of the markers across the gradient (alkaline
phosphodiesterase for the plasma membrane and mannosidase II for Golgi)
(Fig. 1
, G and H), the bulk of cellular protein that corresponded to
Golgi and sarcolemmal membranes was excluded from these fractions
(fractions 46) and equilibrated at the high-sucrose density
(fractions 812) as previously demonstrated (20, 21).

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Figure 1. Subcellular Distribution of Endogenous and
Recombinant Caveolins in COS-7 and Other Cells
Caveolin-enriched membrane fractions were purified on the basis of
their selective resistance to sodium carbonate buffer and their
buoyancy in sucrose density gradients. An aliquot from each of 13
sucrose gradient fractions was subjected to SDS-PAGE. A, Distribution
of total protein after fractionation. Proteins were stained with
Ponceau S. Fractions 18 represent the 535% sucrose layer,
fractions 912 represent the 45% sucrose layer, and fraction 13
represents the insoluble pellet from the cell homogenate of COS-7
cells. Note that the cellular protein remained mainly in the
bottom-loaded sucrose layer (fractions 913). The molecular size
marker is indicated (46, 30, 22 kDa). B, Distribution of endogenous
caveolin-1 in COS-7 cells. Caveolin was detected by immunoblot
analysis. Caveolin migrated on SDS-PAGE with a molecular mass of 24 kDa
and was localized to fractions 5 and 6. The exposure time of the film
was approximately 1 min. C, Distribution of endogenous caveolin-1 in
BAEC. Caveolin in BAEC was similar to that in COS-7 cells. The exposure
time of the film was approximately 1 min. D, Distribution of
recombinant caveolin-1 in COS-7 cells. The recombinant caveolin-1 in
COS-7 cells was detected as double bands (1- and -ß) (36 ) and
migrated slightly slower than the endogenous caveolin-1 (panel B)
because of the myc epitope tag. The exposure time of the film was
approximately 30 sec. E, Distribution of recombinant caveolin-1 in Sf9
cells. Mammalian caveolin-1 was recombinantly expressed in Sf9 insect
cells, which was detected exclusively in fractions 5 and 6. The
exposure time of the film was approximately 30 sec. F, Distribution of
putative endogenous caveolin in Sf9 cells. Immunoblotting with
anticaveolin-1 Ig detected a band migrating with a molecular mass of 30
kDa. Note that the amount of this protein was much smaller than that of
recombinant caveolin-1 (panel E): the exposure time of the film was
approximately 20 min. G and H, Profile of membrane markers across the
gradients in COS-7 cells. Distribution of plasma membrane (alkaline
phosphodiesterase, panel G) and Golgi (mannosidase II, panel H) markers
along sucrose gradient are shown. Numbers 1, 2, and 3 represent
caveolin-enriched fractions (fractions 46), the 40% lower sucrose
layers (fractions 812), and the pellet (fraction 13), respectively.
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Endogenous caveolin-1 was detected by immunoblotting on SDS-PAGE as a
molecular mass of 24 kDa in fractions 46 in COS-7 cells (Fig. 1B
) and
in bovine aortic endothelial cells (BAEC) (Fig. 1C
). Interestingly, the
caveolin antibody detected a protein migrating with an approximate mass
of 30 kDa after long exposure (
20 min) of the film in insect cells
(Fig. 1F
) as well, which was comparable in size to Cavce
(caveolin from Caenorhabditis elegans), a recently
identified invertebrate caveolin (33 kDa) (22). An exogenous,
recombinant caveolin-1 was also detected in these fractions (fractions
46) when overexpressed in COS-7 cells (Fig. 1D
) or in Sf9 insect
cells (Fig. 1E
). These findings suggest that both mammalian and insect
cell systems may be useful to investigate the subcellular localization
of both endogenous and recombinant proteins relative to caveolin.
Adenylyl Cyclase Is Concentrated in the Caveolin-Enriched
Fractions
Caveolin-enriched membrane fractions were isolated from insect
cells overexpressing various adenylyl cyclase isoforms (type III, IV,
and V) (Fig. 2
). These adenylyl cyclase
isoforms were readily detected after subcellular fractionation. They
were found predominantly in the caveolin-enriched membrane fractions
(fractions 46). These findings suggest that the distribution of
adenylyl cyclase within the plasma membrane, in contrast to the random
collision theory, is not consistent throughout the membrane, but
rather is confined to certain subcellular membrane fractions. In
support of our findings, previous cytochemical experiments have
suggested the association of adenylyl cyclase with anatomic structures
resembling caveolae (23, 24). Similarly, Huang et al. (25)
demonstrated the highest specific adenylyl cyclase activity in
caveolin-enriched plasma membrane fractions after detergent-free cell
fractionation.

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Figure 2. Localization of Adenylyl Cyclase in
Caveolin-Enriched Membrane Fractions
Various mammalian adenylyl cyclase isoforms (AC III,
type III; AC IV, type IV; AC V, type V)
were recombinantly expressed in Sf9 cells. Fractions 413 of sucrose
gradients were subjected to immunoblot analysis with isoform-specific
antibodies. Fractions 13 were excluded because they contained little
protein. Isoforms of types III, IV, and V adenylyl cyclase were found
predominantly in the caveolin-enriched membrane fractions. The
molecular size marker (97 kDa) is indicated on each panel.
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More important, since the cloning of multiple adenylyl cyclase
isoforms, numerous laboratories, including our own, have attempted to
identify adenylyl cyclase isoforms by immunoblotting. However, Western
blot analysis of adenylyl cyclase isoforms has been hampered by the low
amount of adenylyl cyclase protein in the plasma membrane and by the
low sensitivity of available antibodies. Our cell fractionation and
purification technique significantly improved the quality of adenylyl
cyclase immunoblots and may be useful for isoform-specific quantitation
of adenylyl cyclase in future studies.
Adenylyl Cyclase Is Detectable in Cardiac Tissue after Sucrose
Gradient Fractionation
Tissue adenylyl cyclase was detected using the above method as
well. We performed the subcellular fractionation of mouse left
ventricular myocardium. Consistent with our findings above, type V
adenylyl cyclase was detected exclusively in the caveolin-enriched
membrane fractions in the mouse heart (Fig. 3A
). This isoform was not detectable when
the crude cardiac homogenate was used (Fig. 3B
, right) but
was detected when the caveolin-enriched membrane fraction was used
(Fig. 3B
, left). Preincubation with the peptide that was
used to raise the antibody blocked the immunostaining, which further
confirmed the specificity of this immunoblotting (Fig. 3C
).

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Figure 3. Immunodetection of Type V Adenylyl Cyclase in
Cardiac Tissues
A, Subcellular distribution of type V adenylyl cyclase
(upper) and caveolin-3 (lower) in the
heart. The homogenate of mouse left ventricular myocardium was
fractionated by sucrose gradient centrifugation, followed by SDS-PAGE
and immunoblotting. Note that type V adenylyl cyclase was detected
exclusively in the caveolin-enriched membrane fractions. B,
Immunoblotting of type V adenylyl cyclase: crude cardiac homogenates
vs. a caveolin-enriched membrane fraction. Sixty
micrograms of either left ventricular homogenate or a caveolin-enriched
membrane fraction were subjected to SDS-PAGE and immunoblotting. Note
that type V adenylyl cyclase was not detectable in the crude homogenate
but became detectable when a caveolin-enriched fraction (fraction 5)
was used. C, Effect of peptide preincubation. Immunoblotting using the
caveolin-enriched membrane fraction and a bottom-loaded fraction are
shown. The antibody was preincubated in the presence (+) and absence
(-) of a 10-fold concentration (relative to that of antiserum
according to the manufactures recommendation) of a synthetic type V
adenylyl cyclase peptide that was used to raise this antibody. The low
molecular masses detected in fractions 11 and 5 were nonspecific since
they were not reproducibly detected in other experiments.
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Copurification of Caveolin with Adenylyl Cyclase
To confirm that caveolin and adenylyl cyclase are physically
bound, we performed copurification experiments using Sf9 cells
coexpressing a polyhistidine epitope-tagged form of type III adenylyl
cyclase and wild-type caveolin-1. The caveolar fraction from the insect
cells was incubated with Ni-nitrilotriacetic acid (NTA) agarose resin,
to which the polyhistidine epitope-tagged type III adenylyl cyclase was
bound. Washing the resin with Tris-buffered saline (TBS) twice and then
three times with TBS/imidazole eliminated nonspecific binding of
caveolin-1. In the absence of a polyhistidine epitope-tagged form of
type III adenylyl cyclase, caveolin-1 was readily washed off from the
resin (Fig. 4
, upper panel).
In the presence of a polyhistidine epitope-tagged form of type III
adenylyl cyclase, however, caveolin-1 was retained on the resin and
detected in the final eluate (Fig. 4
, lower panel). These
data suggest that type III adenylyl cyclase is physically bound to
caveolin-1.

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Figure 4. Copurification of Type III Adenylyl Cyclase with
Caveolin
Caveolin-1 and type III adenylyl cyclase were coexpressed in Sf9 cells.
Caveolin-enriched membrane fractions were obtained by the sucrose
gradient method and incubated with Ni-NTA-agarose resin, which binds to
a polyhistidine epitope-tagged form of type III adenylyl cyclase. After
washing, bound proteins were eluted and subjected to SDS-PAGE, followed
by immunoblotting. Caveolin-1 had a myc epitope at the C terminus.
After washing, bound proteins were eluted and subjected to SDS-PAGE,
followed by immunodetection with a monoclonal antibody to the myc
epitope of caveolin-1. SM (starting material), Fraction 5 of sucrose
gradient before incubation with the resin; RM (remaining material),
supernatant after 6 h incubation with the resin; wash 1 and wash
2, wash with TBS; wash 3-wash 5, wash with TBS containing 30
mM imidazole; eluate, final elution from the resin with TBS
containing 200 mM imidazole. Results from Sf9 cells
overexpressing caveolin-1 alone (upper panel) or both
caveolin-1 and a polyhistidine-tagged form of type III adenylyl cyclase
(lower panel) are shown. Note that caveolin-1 was
detected in the final eluate only in the cells coexpressing type III
adenylyl cyclase with caveolin-1.
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Immunoprecipitation of Adenylyl Cyclase and Caveolin in the
Heart
We also confirmed the association of adenylyl cyclase and caveolin
in cardiac tissues. After homogenization of canine cardiac tissues and
purification of caveolar fractions, caveolin-3 was coimmunoprecipitated
with endogenous type V adenylyl cyclase and Gß (Fig. 5A
). Gß, a membrane-bound protein, is
known to be present in caveolae (26). In contrast, both type V adenylyl
cyclase antibody and Gß were coimmunoprecipitated with caveolin-3 as
well (Fig. 5B
). These results suggest that caveolin-3 forms a complex
with type V adenylyl cyclase in intact tissues.

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Figure 5. Coimmunoprecipitation of Adenylyl Cyclase and
Caveolin in Cardiac Tissues
Caveolar fractions were obtained from canine cardiac tissues after
sucrose gradient centrifugation. In panel A, a Gß antibody or a type
V adenylyl cyclase antibody was used for immunoprecipitation, followed
by immunodetection of caveolin-3 (Cav 3). In panel B, a caveolin-3
antibody was used, followed by immunodetection of type V adenylyl
cyclase (ACV) and Gß (Gß). In control (Control) experiments, the
same concentration of nonimmune serum was used.
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G Proteins Are Diffusely Distributed
The distribution of adenylyl cyclase isoforms differed from that
of heterotrimeric G proteins, which have been shown to be present in
the same membrane fraction as caveolin, but remains controversial in
its exact localization (8, 14, 15, 25). In accordance with these
studies, we found that Gs
was located in caveolin-enriched fractions
and also in the bottom-loaded sucrose layer (Fig. 6A
). The amount in the bottom-loaded
sucrose layer was smaller than that in caveolin-enriched fractions;
however, we consistently detected Gs
in the bottom-loaded sucrose
layer. This diffuse pattern of distribution of Gs
was enhanced when
Gs
was recombinantly overexpressed in COS-7 cells (Fig. 6B
). These
findings suggest that the distribution of Gs
is more diffuse than
that of adenylyl cyclase, which may reflect that G proteins are present
in a large stoichiometric excess over adenylyl cyclase and interact
with multiple other signaling molecules such as Ca channel within the
cell (6).
The Location of PKA Is Subtype Dependent in
Caveolin-Enriched Fractions
We also examined the subcellular localization of PKA subtypes
using cardiac tissues. Immunodetection of PKA was relatively easy in
comparison to that of adenylyl cyclase. As shown in Fig. 7A
, the RII
subunit was enriched in
the caveolar fractions and was also detected in noncaveolar fractions.
In striking contrast, the RI
subunit was excluded from the
caveolin-enriched fractions and was detected in noncaveolar fractions
(Fig. 7B
). We also conducted immunodetection of the catalytic subunit
of PKA, which was detected mostly in noncaveolar fractions but was also
detected in the caveolin-enriched fractions (Fig. 7C
). The distribution
of Gs
(Fig. 7D
) was similar to that in COS-7 cells; Gs
was
located in both caveolin-enriched and noncaveolar fractions (Fig. 6A
).
Caveolin-3, the caveolin subtype in myocytes, was detected only in the
caveolar fractions (Fig. 7E
).
Previous studies have investigated the localization of the two
regulatory subunits of PKA within the cell (27) but not in its relation
to the localization with adenylyl cyclase, which produces cAMP. Our
findings demonstrate that RII
, but not RI
, was concentrated in
the same microdomain as adenylyl cyclase. Thus, the sequential
activation of G proteins and adenylyl cyclase, as well as the
diffusion of cAMP to PKA and its activation, may occur within a
restricted microdomain of the plasma membrane, allowing rapid
transmission of the signal. Our data also suggest that caveolin plays a
role in organizing the membrane localization of these molecules. We do
not know, however, whether caveolin is sufficient or necessary under
any conditions to determine such localization. We also do not know
whether caveolae are made of heterogeneous populations, although a
previous study demonstrated that different caveolin subtypes may
coexist in the same caveolae (28).
The role of caveolae may be more than to colocalize G proteins,
adenylyl cyclase, and PKA. Most recently, we demonstrated that peptides
derived from caveolin subtypes directly inhibited adenylyl cyclase in
an adenylyl cyclase isoform- and a caveolin subtype-dependent manner
(29). Our results are also reminiscent of AKAP79, which colocalizes PKA
and calcineurin (30). It is important to note that AKAP79 inhibits the
activity of calcineurin and PKA (30), suggesting that AKAP79 not only
binds but also regulates signaling molecules.
We did not examine the localization of G protein-coupled receptors in
this study because there are numerous such receptors, and their
subcellular distribution appears to vary. Recent observations
demonstrated the recruitment of B2 bradykinin- and m2 muscarinic
acetylcholine receptors to caveolae in an agonist-regulated fashion
(21, 31) whereas the endothelin receptor subtype A resides in caveolae
under basal conditions as well as after ligand binding (32).
ß-Adrenergic receptors may be located and sequestrated via caveolae
(33, 34) in addition to their redistribution to clathrin-coated pits
upon agonist stimulation (35).
Taken together, our results demonstrate that the molecules involved in
cAMP signaling (G proteins, adenylyl cyclase, and PKA, at least) are
not randomly floating on the lipid membrane of the cell, but are
colocalized in plasma membrane microdomains. The cAMP-signaling pathway
represents a rapid signaling pathway that regulates the function of
multiple organs, such as the heart, that changes on a second-to-second
basis. In this context, preservation of a rapid signaling cascade from
activated G proteins to PKA via diffusible cAMP in the same microdomain
may be of primary importance in maintaining normal function of the
heart. Under pathological conditions such as heart failure, however, it
is unclear whether malfunction of the cAMP-signaling pathway may occur
by disturbing the compartmentation of signaling molecules.
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MATERIALS AND METHODS
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Reagents
Cell Culture Media
Insect Xpress medium was obtained from BioWhittaker, Inc.
(Walkersville, MD). DMEM was from Mediatech Inc. (Herndon, VA).
Antibodies
A polyclonal antibody to Gß was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit serum and rabbit
antimouse serum were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Polyclonal antibodies to
adenylyl cyclase isoforms (types III, IV, and V) as well as a
monoclonal antibody that recognizes the myc epitope of caveolin-1
(9E10) were obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA) (36, 37). A polyclonal antibody to Gs
was obtained
from NEN Life Science Products (Boston, MA). The monoclonal antibody to
the catalytic subunit of PKA, to the C-terminal portion of caveolin-1
(2297), and to caveolin-3 were obtained from Transduction Laboratories, Inc. (Lexington, KY). Polyclonal antibodies to the
RII
- and RI
subunit of PKA were obtained from Chemicon (Temecula,
CA).
cDNA Clones and Plasmids
Caveolin-1 cDNA clone was kindly provided by Dr. M. P. Lisanti.
Type III and IV adenylyl cyclase clones were from Drs. R. R. Reed
and A. G. Gilman, respectively. pBlueBac vector was purchased from
Invitrogen (San Diego, CA).
Other Reagents
Most other reagents were obtained from Sigma Chemical Co.
(St. Louis, MO). LipofectAMINE* was purchased from Life Technologies (Gaithersburg, MD).
Cell Culture
Spodoptera frugiperda (Sf9) insect cells were grown
in Insect Xpress medium containing 6% FBS, penicillin (100 µg/ml),
and streptomycin (100 µg/ml). COS-7 cells and BAEC (kindly provided
by Dr. J. K. Liao) were cultured in DMEM supplemented with 5% or
10% FBS, penicillin, and streptomycin in a humidified 95% air/5%
CO2 incubator.
Membrane Marker Assays
Markers of the plasma membrane (alkaline phosphodiesterase) and
Golgi (mannosidase II) were assayed as described previously (21, 38).
In brief, alkaline phosphodiesterase and mannosidase II activities were
determined by hydrolysis of thymidine 5-monophosphate
p-nitrophenyl ester or p-nitrophenyl
-D-mannopyranoside, respectively. After incubation for
2 h (alkaline phosphodiesterase) or 1 h (mannosidase II) at
37 C, absorbance was measured at 405 nm using a microplate reader
(EG&G, Gaithersburg, MD).
Transfection of COS-7 Cells
COS-7 cells were plated in 10-cm dishes at approximately 60%
confluence. For transfection, 16 µl LipofectAMINE* were mixed with 8
µg caveolin-1 cDNA in serum- and antibiotic-free DMEM. After 30 min,
the LipofectAMINE*/cDNA solution was diluted with 4.8 ml DMEM
supplemented with 5% FBS and overlaid onto the cells. After 6 h
incubation at 37 C in 5% CO2, the solution was replaced
with complete DMEM culture medium containing 5% FBS and antibiotics.
The cells were collected for experiments 48 h after
transfection.
Overexpression of Adenylyl Cyclase and Caveolin-1 in Insect
Cells
Adenylyl cyclase isoforms (type III, IV, and V) were
overexpressed in insect cells as previously described (39, 40, 41). A
XhoI/KpnI fragment from the caveolin-1 cDNA
containing a myc epitope was used as previously described (36). These
clones were inserted into the pBlueBac vector. The recombinant shuttle
vectors were transfected into insect cells using the Bac-N-Blue
transfection kit. The plaques were then purified as previously
described (39). Forty-eight hours after infection, cells were washed
twice with ice-cold PBS (137 mM NaCl, 2.7 mM
KCl, 4.3 mM Na2HPO4, 1.4
mM KH2PO4, pH 7.5) and subjected to
sucrose gradient centrifugation.
Cell Fractionation by Sucrose Gradient Centrifugation
Caveolin-enriched membrane domains were purified from cultured
insect cells, mammalian cells, and tissues by a previously optimized
method (17). All steps were carried out at 4 C. Cells were resuspended
in 2 ml of 500 mM sodium carbonate (pH 11). Homogenization
of the suspension with 12 strokes of a Dounce homogenizer was followed
by three 10-sec bursts of a Polytron tissue grinder and four 20-sec
bursts of a sonicator. In experiments starting from tissue and isolated
cardiomyocytes, nine 20-sec bursts of a sonicator were performed. The
sucrose concentration in cell extracts was adjusted to 45% by the
addition of 2 ml of 90% sucrose prepared in MBS [25 mM
4-morpholinoethanesulfonic acid (MES), pH 6.5; 0.15 M
NaCl], and the extracts were placed at the bottom of an
ultracentrifuge tube. A 535% discontinuous sucrose gradient was
formed above (4 ml of 35%/4 ml of 5% sucrose, both prepared in MBS
containing 250 mM sodium carbonate), and centrifuged at
39,000 rpm for 16 h at 4 C in a Sorvall TH 641 rotor. A
light-scattering band was confined to the 535% sucrose interface.
From the top of each gradient, a total of 13 fractions (1 ml each) were
collected.
Immunoblotting
Gradient fractions were separated by SDS-PAGE and transferred to
Immobilon-P membranes (Millipore Corp., Bedford, MA).
After transfer, the membranes were blocked in 5% nonfat dry milk and
subjected to immunoblotting. Bound primary antibodies were visualized
using Amersham (Arlington Heights, IL) or Pierce Chemical Co. (Rockford, IL) chemiluminescence Western blotting
detection reagents.
Affinity Chromatography
The caveolin-enriched fraction (fraction 5) from Sf9
insect cells overexpressing caveolin-1 and a polyhistidine-tagged form
of adenylyl cyclase type III was adjusted to pH 8 using 1 M
MES buffer, followed by incubation with Ni-NTA-agarose resin
(Qiagen, Chatsworth, CA) for 6 h at 4 C (17). The
Ni-NTA-agarose resin was allowed to settle by gravity (5 min on ice)
and washed twice with TBS (10 mM Tris, pH 8, and 0.15
M NaCl) and then three times with TBS containing 30
mM imidazole (5 min each). Finally, bound proteins were
eluted with TBS containing 200 mM imidazole for 1 h
at 4 C. Washes and eluates were subjected to immunoblot
analysis.
Immunoprecipitation
Canine cardiac tissues were homogenized and fractionated to
obtain caveolar fractions. Fractions 46 were collected. After
dilution (1:2, vol/vol) in buffer A (50 mM Tris-HCl, pH
8.0, 2 mM EGTA, 1 mM dithiothreitol, and
protease inhibitors), they were pelleted by centrifugation at
100,000 x g for 30 min. The pellets were lysed in buffer B (50
mM Tris-HCl, pH 7.4, 60 mM ß-octyglucoside,
10 mM EDTA, 1% Nonidet p-40, 0.4% deoxycholate, and
protease inhibitors). The lysates were incubated with a specific
antibody (anti-type V adenylyl cyclase or Gß polyclonal antibody or
anticaveolin-3 monoclonal antibody) for 12 h at 4 C. In the case
of antimonoclonal antibody, the rabbit antimouse antibody was also
added. Immune complexes formed by the addition of protein A-Sepharose
were incubated for 2 h at 4 C. The immune complexes were then
sedimented by centrifugation at 14,000 rpm, followed by washing with
3 x 1 ml of ice-cold buffer C (25 mM MES, 150
mM NaCl, 1% Triton X-100). Bound proteins were solubilized
and analyzed on SDS-PAGE, followed by immunoblotting.
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FOOTNOTES
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Address requests for reprints to: Yoshihiro Ishikawa, Cardiovascular & Pulmonary Research Institute, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212.
This study was supported by grants from the United States Public Health
Service (HL-38070 and HL-59729), the American Heart Association (Grant
9940187), and the Uehara Memorial Foundation. C.S. was supported by the
Deutsche Forschungsgemeinschaft. Y.I. is a recipient of the Established
Investigator Award from the American Heart Association.
Received for publication June 10, 1998.
Revision received February 23, 1999.
Accepted for publication February 25, 1999.
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