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Cardiovascular & Pulmonary Research Institute Allegheny University of the Health Sciences Pittsburgh, Pennsylvania 15212
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 Ross’s 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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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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 4–6) and equilibrated at the high-sucrose density
(fractions 8–12) as previously demonstrated (20, 21).
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) 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
4–6) 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 4–6). 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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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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, 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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). 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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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).
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, 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).
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, 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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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 5–35% 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 5–35% 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 4–6 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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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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q and Gi in
caveolae in DDT1 MF-2 smooth muscle cells. J Biol Chem 272:17858–17866 subunits and ß subunits of
heterotrimeric G proteins on Golgi membranes of the exocrine pancreas.
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 N. Defer, M. Best-Belpomme, and J. Hanoune Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase Am J Physiol Renal Physiol, September 1, 2000; 279(3): F400 - F416. [Abstract] [Full Text] [PDF]
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 Q. He, G. Wu, and M. C. Lapointe Isoproterenol and cAMP regulation of the human brain natriuretic peptide gene involves Src and Rac Am J Physiol Endocrinol Metab, June 1, 2000; 278(6): E1115 - E1123. [Abstract] [Full Text] [PDF]
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 J. M. Hare, R. A. Lofthouse, G. J. Juang, L. Colman, K. M. Ricker, B. Kim, H. Senzaki, S. Cao, R. S. Tunin, and D. A. Kass Contribution of Caveolin Protein Abundance to Augmented Nitric Oxide Signaling in Conscious Dogs With Pacing-Induced Heart Failure Circ. Res., May 26, 2000; 86(10): 1085 - 1092. [Abstract] [Full Text] [PDF]
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R. S. Ostrom, J. D. Violin, S. Coleman, and P. A. Insel Selective Enhancement of beta -Adrenergic Receptor Signaling by Overexpression of Adenylyl Cyclase Type 6: Colocalization of Receptor and Adenylyl Cyclase in Caveolae of Cardiac Myocytes Mol. Pharmacol., May 1, 2000; 57(5): 1075 - 1079. [Abstract] [Full Text]
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K. A. Fagan, K. E. Smith, and D. M. F. Cooper Regulation of the Ca2+-inhibitable Adenylyl Cyclase Type VI by Capacitative Ca2+ Entry Requires Localization in Cholesterol-rich Domains J. Biol. Chem., August 18, 2000; 275(34): 26530 - 26537. [Abstract] [Full Text] [PDF]
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V. O. Rybin, X. Xu, M. P. Lisanti, and S. F. Steinberg Differential Targeting of beta -Adrenergic Receptor Subtypes and Adenylyl Cyclase to Cardiomyocyte Caveolae. A MECHANISM TO FUNCTIONALLY REGULATE THE cAMP SIGNALING PATHWAY J. Biol. Chem., December 22, 2000; 275(52): 41447 - 41457. [Abstract] [Full Text] [PDF]
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B. Razani, T. P. Combs, X. B. Wang, P. G. Frank, D. S. Park, R. G. Russell, M. Li, B. Tang, L. A. Jelicks, P. E. Scherer, et al. Caveolin-1-deficient Mice Are Lean, Resistant to Diet-induced Obesity, and Show Hypertriglyceridemia with Adipocyte Abnormalities J. Biol. Chem., March 1, 2002; 277(10): 8635 - 8647. [Abstract] [Full Text] [PDF]
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E. L. Kilpatrick, P. Narayan, R. M. Mentzer Jr., and R. D. Lasley Cardiac myocyte adenosine A2a receptor activation fails to alter cAMP or contractility: role of receptor localization Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H1035 - H1040. [Abstract] [Full Text] [PDF]
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