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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, but not RI{alpha}) was also enriched in the same fraction as caveolin. Gs{alpha} 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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{alpha} and -ß, caveolin-2 and -3) that differ in tissue distribution (14). The recent identification of various G protein {alpha}- and ß{gamma}-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{alpha} (reviewed in Refs. 1, 18).


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). 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. 1Go, 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).



View larger version (42K):
[in this window]
[in a new window]
 
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 1–8 represent the 5–35% sucrose layer, fractions 9–12 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 9–13). 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-{alpha} 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 4–6), the 40% lower sucrose layers (fractions 8–12), and the pellet (fraction 13), respectively.

 
Endogenous caveolin-1 was detected by immunoblotting on SDS-PAGE as a molecular mass of 24 kDa in fractions 4–6 in COS-7 cells (Fig. 1BGo) and in bovine aortic endothelial cells (BAEC) (Fig. 1CGo). 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. 1FGo) 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. 1DGo) or in Sf9 insect cells (Fig. 1EGo). 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. 2Go). 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.



View larger version (44K):
[in this window]
[in a new window]
 
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 4–13 of sucrose gradients were subjected to immunoblot analysis with isoform-specific antibodies. Fractions 1–3 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.

 
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. 3AGo). This isoform was not detectable when the crude cardiac homogenate was used (Fig. 3BGo, right) but was detected when the caveolin-enriched membrane fraction was used (Fig. 3BGo, left). Preincubation with the peptide that was used to raise the antibody blocked the immunostaining, which further confirmed the specificity of this immunoblotting (Fig. 3CGo).



View larger version (41K):
[in this window]
[in a new window]
 
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 manufacture’s 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.

 
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. 4Go, 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. 4Go, lower panel). These data suggest that type III adenylyl cyclase is physically bound to caveolin-1.



View larger version (56K):
[in this window]
[in a new window]
 
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.

 
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. 5AGo). 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. 5BGo). These results suggest that caveolin-3 forms a complex with type V adenylyl cyclase in intact tissues.



View larger version (25K):
[in this window]
[in a new window]
 
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.

 
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{alpha} was located in caveolin-enriched fractions and also in the bottom-loaded sucrose layer (Fig. 6AGo). The amount in the bottom-loaded sucrose layer was smaller than that in caveolin-enriched fractions; however, we consistently detected Gs{alpha} in the bottom-loaded sucrose layer. This diffuse pattern of distribution of Gs{alpha} was enhanced when Gs{alpha} was recombinantly overexpressed in COS-7 cells (Fig. 6BGo). These findings suggest that the distribution of Gs{alpha} 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).



View larger version (39K):
[in this window]
[in a new window]
 
Figure 6. Subcellular Distribution of Endogenous and Recombinant Gs{alpha}

A, Subcellular distribution of endogenous Gs{alpha} in COS-7 cells. After cell fractionation, endogenous Gs{alpha} was detected by immunoblotting. Note that both Gs{alpha} subtypes (45 and 52 kDa) were detected in fractions 4–6 as well as in fractions 9–12. B, Subcellular distribution of recombinant Gs{alpha} in COS-7 cells. A larger 52-kDa Gs{alpha} was recombinantly overexpressed in COS-7 cells, followed by cell fractionation and immunoblotting. Note that the 52-kDa Gs{alpha} was detected mostly in fractions 9–12.

 
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. 7AGo, the RII{alpha} subunit was enriched in the caveolar fractions and was also detected in noncaveolar fractions. In striking contrast, the RI{alpha} subunit was excluded from the caveolin-enriched fractions and was detected in noncaveolar fractions (Fig. 7BGo). 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. 7CGo). The distribution of Gs{alpha} (Fig. 7DGo) was similar to that in COS-7 cells; Gs{alpha} was located in both caveolin-enriched and noncaveolar fractions (Fig. 6AGo). Caveolin-3, the caveolin subtype in myocytes, was detected only in the caveolar fractions (Fig. 7EGo).



View larger version (50K):
[in this window]
[in a new window]
 
Figure 7. Subcellular Distribution of PKA Subunits

Left ventricular tissues were subjected to subcellular fractionation. Fractions 4–13 were immunoblotted with specific antibodies to the RII{alpha} subunit (panel A), the RI{alpha} subunit (panel B), the catalytic subunit (panel C), Gs{alpha} (panel D), and caveolin-3 (panel E).

 
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{alpha}, but not RI{alpha}, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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{alpha}- and RI{alpha} 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 {alpha}-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.


    FOOTNOTES
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ishikawa Y, Homcy CJ 1997 The adenylyl cyclases as integrators of transmembrane signal transduction. Circ Res 80:297–304[Free Full Text]
  2. Scott JD 1991 Cyclic nucleotide-dependent protein kinases. Pharmacol Ther 50:123–145[CrossRef][Medline]
  3. Orly J, Schramm M 1976 Coupling of catecholamine receptor from one cell with adenylyl cyclase from another cell by cell fusion. Proc Natl Acad Sci USA 73:4410–4414[Abstract]
  4. Tolkovsky AM, Levitzki A 1978 Model of coupling between the ß-adrenergic receptor and adenylyl cyclase in turkey erythrocytes. Biochemistry 17:3795–3810[Medline]
  5. Pawson T, Scott JD 1997 Signaling through scaffold, anchoring, and adapter proteins. Science 278:2075–2080[Abstract/Free Full Text]
  6. Neubig RR 1994 Membrane organization in G protein mechanism. FASEB J 8:939–946[Abstract/Free Full Text]
  7. Houslay MD, Milligan G 1997 Tailoring cAMP-signaling responses through isoform multiplicity. Trends Biochem Sci 22:189–232[CrossRef][Medline]
  8. Venter JC, Ross JJ, Kaplan NO 1975 Lack of detectable change in cyclic AMP during the cardiac inotropic response to isoproterenol immobilized on glass beads. Proc Natl Acad Sci USA 72:824–828[Abstract]
  9. Pedersen SE, Ross EM 1982 Functional reconstitution of beta-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase. Proc Natl Acad Sci USA 79:7228–7232[Abstract]
  10. Jurevicius J, Fischmeister R 1996 cAMP compartmentation is responsible for a local activation of cardiac Ca channels by ß-adrenergic agonists. Proc Natl Acad Sci USA 93:295–299[Abstract/Free Full Text]
  11. Fagan KA, Mahey R, Cooper DMF 1996 Functional co-localization of transfected Ca-stimulable adenylyl cyclases with capacitative Ca entry sites. J Biol Chem 271:12438–12444[Abstract/Free Full Text]
  12. Simionescu N, Simionescu M, Palade GE 1972 Permeability of intestinal capillaries. J Cell Biol 53:365–392[Abstract/Free Full Text]
  13. Anderson RGW 1993 Plasmalemmal caveolae and GPI-anchored membrane proteins. Curr Opin Cell Biol 5:647–652[Medline]
  14. Couet J, Li S, Okamoto T, Scherer PE, Lisanti MP 1997 Molecular and cellular biology of caveolae. Trends Cardiovasc Med 7:103–110[CrossRef]
  15. Chang W-J, Ying J-S, Rothberg KG, Hooper NM, Turner AJ, Gambliel HA, De Gunzburg J, Mumby SM, Gilman AG, Anderson RGW 1994 Purification and characterization of smooth muscle cell caveolae. J Cell Biol 126:127–138[Abstract]
  16. Li S, Okamoto T, Chun M, Sargiacomo M, Casanova JE, Hansen SH, Nishimoto I, Lisanti MP 1995 Evidence for a regulated interaction between heterotrimeric G proteins and caveolin. J Biol Chem 270:15693–15701[Abstract/Free Full Text]
  17. Song KE, Li S, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti M 1996 Co-purification and direct interaction of ras with caveolin, an integral membrane protein of caveolae microdomains. J Biol Chem 271:9690–9697[Abstract/Free Full Text]
  18. Sunahara RK, Dessauer KW, Gilman AG 1996 Complexity and diversity of mammalian adenylyl cyclases. Annu Rev Pharmacol Toxicol 36:461–480[CrossRef][Medline]
  19. Sargiacomo M, Sudol M, Tang ZL, Lisanti MP 1993 Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol 122:789–807[Abstract]
  20. Lisanti MP, Scherer PE, Vidugiriene J, Tang ZL, Hermanowski-Vosatka A, Tu YH, Cook RF, Sargiacomo M 1994 Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implication for human disease. J Cell Biol 126:111–126[Abstract]
  21. Feron O, Smith TW, Michel T, Kelly RA 1997 Dynamic targeting of the agonist-stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes. J Biol Chem 272:17744–17748[Abstract/Free Full Text]
  22. Tang Z-L, Okamoto T, Boontrakulpoontawee P, Otsuka AJ, Lisanti MP 1997 Identification, sequence, and expression of an invertebrate caveolin gene family from the nematode Caenorhabditis elegans. J Biol Chem 272:2437–2445[Abstract/Free Full Text]
  23. Wagner RC, Kreiner P, Barrnett RJ, Bitensky MW 1972 Biochemical characterization and cytochemical localization of a catecholamine sensitive adenylate cyclase in isolated capillary endothelium. Proc Natl Acad Sci USA 69:3175–3179[Abstract]
  24. Slezak J, Geller SA 1984 Cytochemical studies of myocardial adenylate cyclase after its activation and inhibition. J Histochem Cytochem 32:105–113[Abstract]
  25. Huang C, Hepler JR, Chen LT, Gilman AG, Anderson RGW, Mumby SM 1997 Organization of G proteins and adenylyl cyclase at the plasma membrane. Mol Biol Cell 8:2365–2378[Abstract/Free Full Text]
  26. Song KS, Scherer PE, Tang Z-L, Okamoto T, Li S, Chafel M, Chu C, Kohtz DS, Lisanti MP 1996 Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. J Biol Chem 271:15160–15165[Abstract/Free Full Text]
  27. Mochly-Rosen D 1995 Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 268:247–251[Medline]
  28. Scherer PE, Lewis RV, Volont D, Engelman JA, Galbiati F, Couet J, Kohtz DS, van Donselaar E, Peters P, Lisanti MP 1997 Cell-type and tissue-specific expression of caveolin-2: caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J Biol Chem 272:29337–29346[Abstract/Free Full Text]
  29. Toya Y, Schwencke C, Couet J, Lisanti MP, Ishikawa Y 1998 Inhibition of adenylyl cyclase by caveolin. Endocrinology 139:2025–2031[Abstract/Free Full Text]
  30. Coghlan VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM, Scott JD 1995 Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267:108–111[Medline]
  31. de Weerd WFC, Leeb-Lundberg LMF 1997 Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled G{alpha}q and G{alpha}i in caveolae in DDT1 MF-2 smooth muscle cells. J Biol Chem 272:17858–17866[Abstract/Free Full Text]
  32. Chun M, Liyanage UK, Lisanti MP, Lodish HF 1994 Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolae. Proc Natl Acad Sci USA 91:11728–11732[Abstract/Free Full Text]
  33. Raposo G, Dunia I, Delavier-Klutchko C, Kaveri S, Strosberg AD, Benedeth EL 1989 Internalization of the ß-adrenergic receptor in A431 cells involves non-coated vesicles. Eur J Cell Biol 50:340–352[Medline]
  34. Dupree P, Parton RG, Raposo G, Kurzchalia TV, Simons K 1993 Caveolae and sorting in the trans-Golgi network of epithelial cells. EMBO J 12:1597–1605[Abstract]
  35. Kallal L, Gagnon AW, Penn RB, Benovic JB 1998 Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged ß2-adrenergic receptor. J Biol Chem 273:322–328[Abstract/Free Full Text]
  36. Scherer PE, Tang Z, Chun M, Sargiacomo M, Lodish HF, Lisanti MP 1995 Caveolin isoforms differ in their N-terminal protein sequence and subcellular distribution. Identification and epitope mapping of an isoform-specific monoclonal antibody probe. J Biol Chem 270:16395–16401[Abstract/Free Full Text]
  37. Wei J, Wayman G, Storm DR 1996 Phosphorylation and inhibition of type III adenylyl cyclase by calmodulin-dependent protein kinase II in vivo. J Biol Chem 271:24231–24235[Abstract/Free Full Text]
  38. Denker SP, McCaffery JM, Palade GE, Farquhar MG 1996 Differential distribution of {alpha} subunits and ß{gamma} subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas. J Cell Biol 133:1027–1040[Abstract]
  39. Kawabe J, Ebina T, Ismail S, Kitchen D, Homcy CJ, Ishikawa Y 1994 A novel peptide inhibitor of adenylyl cyclase (AC): a peptide from type V AC directly inhibits AC catalytic activity. J Biol Chem 269:24906–24911[Abstract/Free Full Text]
  40. Ebina T, Toya Y, Oka N, Kawabe J-I, Schwencke C, Ishikawa Y 1997 Isoform-dependent activation of adenylyl cyclase by proteolysis. FEBS Lett 401:223–226[CrossRef][Medline]
  41. Ebina T, Toya Y, Oka N, Schwencke C, Kawabe J, Ishikawa Y 1997 Isoform-specific regulation of adenylyl cyclase by oxidized catecholamine. J Mol Cell Cardiol 29:1247–1254[CrossRef][Medline]