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Endocrinology Vol. 139, No. 4 2025-2031
Copyright © 1998 by The Endocrine Society

ARTICLES

Inhibition of Adenylyl Cyclase by Caveolin Peptides1

Yoshiyuki Toya, Carsten Schwencke, Jacques Couet, Michael P. Lisanti and Yoshihiro Ishikawa

Cardiovascular and Pulmonary Research Institute (Y.T., C.S., Y.I.), Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212; and Department of Molecular Pharmacology (J.C., M.P.L.), Albert Einstein Medical College, New York, New York 10461

Address all correspondence and requests for reprints to: Yoshihiro Ishikawa, M.D., Ph.D., Cardiovascular and Pulmonary Research Institute, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212. E-mail: yishikaw{at}pgh.auhs.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Caveolae and their principal component caveolin have been implicated in playing a major role in G protein-mediated transmembrane signaling. We examined whether caveolin interacts with adenylyl cyclase, an effector of G protein signaling, using a 20-mer peptide derived from the N-terminus scaffolding domain of caveolin-1. When tissue adenylyl cyclases were examined, cardiac adenylyl cyclase was inhibited more potently than other tissue adenylyl cyclases. The caveolin-1 peptide inhibited type V, as well as type III adenylyl cyclase, overexpressed in insect cells, whereas the same peptide had no effect on type II. The caveolin-3 scaffolding domain peptide similarly inhibited type V adenylyl cyclase. In contrast, peptides derived from the caveolin-2 scaffolding domain and a caveolin-1 nonscaffolding domain had no effect. Kinetic studies showed that the caveolin-1 peptide decreased the maximal rate (Vmax) value of type V without changing the Michaelis constant (Km) value for the substrate ATP. Studies with various truncations and point mutations of this peptide revealed that a minimum of 16 amino acid residues and intact aromatic residues are important for the inhibitory effect. The potency of inhibition was greater when adenylyl cyclase was in stimulated condition vs. basal condition. Thus, caveolin may be another cellular component that regulates adenylyl cyclase catalytic activity. Our results also suggest that the caveolin peptide may be used as an isoform-selective inhibitor of adenylyl cyclase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CAVEOLIN is a major protein component of caveolae, flask-shaped plasma membrane invaginations, that exist in most cell types, including endothelial cells, fibroblasts, adipocytes, and myocytes (1, 2, 3, 4, 5, 6). There are multiple members of the caveolin gene family (at least three subtypes) that differ in tissue distribution (6, 7, 8, 9), although the significance of this distinct tissue distribution and putative functional diversity remain poorly understood.

Caveolae may participate in transmembrane signaling, in particular, G protein-coupled signaling (5). Histochemical studies demonstrated the enrichment of various G protein-coupled receptors, including the muscarinic receptor and the ß-adrenergic receptor, in caveolae (10, 11). The ß-adrenergic receptor may internalize via caveolae (10, 12). Various G proteins (Go, Gs, Gi, Gq, and Gß{gamma}) and G protein related toxins (cholera and tetanus toxins) have also been demonstrated to be enriched in caveolae by both histochemical and biochemical methods (6, 13, 14, 15).

Furthermore, caveolin may directly interact with molecules that are accumulated in caveolae and regulate their function. Different groups have demonstrated the interaction of caveolin with G proteins, Src kinases, Ha-Ras, and nitric oxide synthase, which are all enriched in caveolae, using a short stretch of membrane proximal regions of the cytosolic amino-terminus domain of caveolin (or the caveolin scaffolding domain) (9, 14, 16, 17, 18). Further, a 20-mer peptide derived from this domain bound G-protein directly and regulated its function (19).

We have recently found that adenylyl cyclase, an effector enzyme of G protein-coupled signaling, is highly enriched in the caveolae fraction (20), which has prompted us to investigate the functional interaction of caveolin with adenylyl cyclase. We demonstrate the caveolin-mediated regulation of adenylyl cyclase catalytic activity by the use of the caveolin scaffolding domain peptides. We also demonstrate that this regulation occurs differently among caveolin subtypes, adenylyl cyclase isoforms, and various tissue adenylyl cyclases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptide synthesis
Caveolin peptides were prepared as previously described (19). The following four peptides were used most often; a caveolin-1 nonscaffolding domain peptide (NRDPKHLNDDVVKIDFEDVIAEPEGTHSF, caveolin-1 amino acid residues 53–81); the caveolin-1 scaffolding domain peptide (DGIWKASFTTFTVTKYWFYR, amino acid residues 82–101); the caveolin-2 scaffolding domain peptide (DKVWICSHALFEISKYVMYK, amino acid residues 54- 73); and the caveolin-3 scaffolding domain peptide (DGVWRVSYTTFTVSKYWCYR, amino acid residues 55–74). The peptides were made by Research Genetics (Huntsville, AL) or Bio Synthesis (Lewisville, TX). Peptides were dissolved in 100% dimethylsulfoxide and diluted to a final concentration of 1.25–10 µM at the time of assay. The final concentration of dimethylsulfoxide was kept constant among assay tubes below 2%, which did not affect catalytic activity.

The proportion of the identity in the corresponding amino acid residue among the caveolin scaffolding domain peptides is 35% (between caveolin 1 and 2), 45% (between caveolin 2 and 3), and 70% (between caveolin 1 and 3). Similarity is 55% (between caveolin 1 and 2), 60% (between caveolin 2 and 3), and 95% (between caveolin 1 and 3). The overall identity among the three is 35%, and similarly is 55%.

Tissue preparations
Mouse tissues (heart, brain, and lung) were minced in a buffer containing 50 mM Tris/HCl (pH 8.0), 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 200 mM sucrose, and a protease inhibitor mixture (10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 U/ml egg white trypsin inhibitor, 20 µg/ml L-1-tosylamide-2-phenylethyl chloromethyl ketone, and 2 µg/ml aprotinin). The tissues were then homogenized with a Polytron for 3 x 10 seconds, followed by centrifugation at 500 x g for 10 min at 4 C. The supernatants were retained and further centrifuged at 100,000 x g for 40 min at 4 C. The crude membrane preparations were made by resuspending the pellet in the same buffer without EGTA and were stored at -80 C until use. The animals were maintained in accordance with guidelines of the Institutional Animal Care and Use Committee of the Allegheny University of the Health Sciences.

Overexpression of adenylyl cyclase
Adenylyl cyclase isoforms of types II and III (a generous gift from Dr. R. R. Reed, The Johns Hopkins University, Baltimore, MD), and type V (21) were overexpressed in High Five (H5) insect cells, as previously described (22, 23, 24, 25). Briefly, insect cells were infected with recombinant baculovirus and incubated in insect media (Insect X-press, BioWhittaker, Walkersville, MD), containing 5% FBS, at 27 C for 3 days before harvesting.

HEK293 cells overexpressing type V adenylyl cyclase (26) were grown at 37 C in DMEM, supplemented with 5% FBS, in a humidified 95% air-5% CO2 incubator.

Cell membrane preparations
Insect and HEK293 cells were washed twice with ice-cold PBS and homogenized in a buffer containing 50 mM Tris/HCl (pH 8.0), 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 200 mM sucrose, and a protease inhibitor mixture containing 20 µg/ml 1-chloro-3-tosylamido-7-amino-L-2-heptanone, 10 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 50 U/ml egg white trypsin inhibitor, and 2 µg/ml aprotinin (22). Cells were disrupted with a sonicator and centrifuged at 500 x g for 10 min at 4 C. The supernatants were further centrifuged at 100,000 x g for 40 min at 4 C. The resultant pellets were resuspended in the same buffer without EGTA. The crude membrane preparations were stored at -80 C until use.

Adenylyl cyclase assay
Adenylyl cyclase activity was measured as previously described (23). The reaction mixtures contained 20 mM HEPES (pH 8.0), 5 mM MgCl2, 0.5 mM EDTA, 0.2 mM ATP containing {alpha}-32P-ATP (1 x 106 cpm), 0.2 mM cAMP containing 3H-cAMP (1 x 104 cpm), 1 mM creatine phosphate, 8 U/ml creatine phosphokinase, 0.5 mM 1-methyl-3-isobutylxanthine, and 2–5 µg of the membrane protein in a final vol of 100 µl. The assay was performed at 30 C for 20 min and terminated by the addition of 100 µl of ice-cold 2% SDS, followed by heating at 80 C for 10 min. cAMP formed during the incubation was separated with Dowex and neutral alumina columns (27) and corrected for recovery with 3H-cAMP. Protein concentration was measured with the Bio-Rad protein assay system (Bio-Rad Laboratories, Hercules, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of caveolin peptide on tissue adenylyl cyclases
We first examined whether caveolin regulates the catalytic activity of adenylyl cyclase. We overexpressed a polyhistidine-tagged form of caveolin-1 in insect cells and partially purified it using affinity chromatography (Ni-NTA agarose resin). The purified caveolin-1 seemed to inhibit adenylyl cyclase; however, we could not demonstrate the inhibition convincingly because caveolin-1 was aggregated at high concentrations. Aggregation of caveolin in the native condition has been reported previously (28, 29).

To avoid the aggregation problem, we used instead a peptide derived from the caveolin-1 scaffolding domain (amino acid residues 82–101), which was previously demonstrated to interact with G proteins (14, 17). We examined whether the caveolin peptide regulated the catalytic activity of tissue adenylyl cyclases (Fig. 1Go).



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  		Figure 1. Effect of the caveolin-1 peptide on tissue adenylyl cyclases. The effect of the caveolin-1 scaffolding domain peptide (0–10 µM) was examined on various tissue adenylyl cyclases obtained from mice (closed circles, heart; open circles, brain; closed triangles, lung). Assays were performed in the presence of 50 µM forskolin. Adenylyl cyclase catalytic activity was expressed as percentage of the value without the peptide. Means ± SEM for four independent experiments are shown. The absolute value for forskolin-stimulated activity was 1,856 pmol/min·mg in the brain, 164.2 pmol/min·mg in the heart, and 132.5 pmol/min·mg in the lung. For comparison, basal activity was 152.2 pmol/min·mg in the brain, 12.1 pmol/min·mg in the heart, and 14.2 pmol/min·mg in the lung (average of three independent determinations).

 
Tissue homogenates were obtained from the heart, brain, and lung of mice. The catalytic activity of brain adenylyl cyclase was 10-fold greater than that of the other tissue adenylyl cyclases. All tissue adenylyl cyclases were inhibited by the caveolin peptide dose-dependently (Fig. 1Go). The inhibitory effect of the caveolin-1 peptide was greatest on cardiac adenylyl cyclase but modest on brain and lung adenylyl cyclases. These findings suggested that the sensitivity of each tissue adenylyl cyclase to the caveolin peptide was different. Because each tissue expresses a distinctive mixture of multiple adenylyl cyclase isoforms (30), each adenylyl cyclase isoform may have different sensitivity to the caveolin peptide. For example, the heart expresses type V as the major isoform and other isoforms, such as types IV, VI, and VII; this is different in the lung, in which type II is expressed as the major isoform (21, 30, 31, 32, 33, 34).

Inhibition of the adenylyl cyclase isoform by the caveolin peptide
Because of the lack of antibodies that can quantitate tissue adenylyl cyclase isoforms with a high sensitivity, we could not determine which adenylyl cyclase isoforms were inhibited by the caveolin peptide in the above study. Thus, we examined the effect of the caveolin peptide on the recombinant adenylyl cyclase isoform overexpressed in insect cells as the dominant positive isoform. The overexpression of the recombinant adenylyl cyclase isoform increased catalytic activity of the insect cell membrane by approximately 10-fold (for type II), 7-fold (for type III), and 50-fold (for type V), as determined in the presence of forskolin.

The caveolin-1 scaffolding domain peptide potently inhibited type V adenylyl cyclase (Fig. 2Go). In our preliminary study, the inhibitory effect of caveolin peptide was much greater on adenylyl cyclase in forskolin-stimulated condition (by 90%) than in basal condition (by 55%). Thus, we used forskolin-stimulated condition in the following experiments. The caveolin peptide from different lots or the peptide provided by different manufacturers showed similar effect.



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  		Figure 2. Dose-response effect of the caveolin-1 peptide on adenylyl cyclase catalytic activity. The adenylyl cyclase isoforms (open circles, ACII, type II; closed circles, ACIII, type III; triangles, ACV, type V) overexpressed in H5 insect cells were incubated in the presence of an increasing concentration of the caveolin-1 scaffolding domain peptide (amino acid residues 82–101) (Peptide) and 50 µM forskolin for assays. All assays were performed in duplicate, and the results are shown as means ± SEM from four independent experiments. *, P < 0.05; **, P < 0.01, from the control values. Inset, The results are also presented as percentage of the values without the peptide.

 
We then examined the effect of this peptide on the other adenylyl cyclase isoforms (types II and III) to exclude that the inhibition was nonspecific and, more important, to test whether the peptide had any isoform selectivity of inhibition because the degree of inhibition was different among tissues in the above study (Fig. 1Go). Further, isoform-selective inhibition of adenylyl cyclase is well known with Gß{gamma} and Gi{alpha} (35). As shown in Fig. 2Go, the peptide inhibited type III similarly, as did type V. In contrast, the peptide had no effect on type II, at least in the range of concentrations that we examined (1–10 µM).

Catalytic activity, in the absence of the peptide, was highest for type V and lowest for type III. Thus, it was unlikely that the inhibitory effect was simply proportional or inversely proportional to the catalytic activity of each isoform. These findings suggested that the interaction of the caveolin peptide and adenylyl cyclase occurred in an adenylyl cyclase isoform-dependent manner.

Effect of other caveolin peptides
To examine whether type V adenylyl cyclase was inhibited by any caveolin peptides, we determined the effect of peptides derived from the scaffolding domain of caveolin-2 (amino acid residues 55–73) and -3 (amino acid residues 55–73). We also examined a peptide from the nonscaffolding domain of caveolin-1 (amino acid residues 53–81) to examine whether any caveolin-1 peptides could inhibit adenylyl cyclase. As shown in Fig. 3Go, the caveolin-3 scaffolding domain peptide inhibited adenylyl cyclase to the same degree as the caveolin-1 peptide, whereas the caveolin-2 scaffolding domain peptide and the caveolin-1 nonscaffolding domain peptide had no effect. These data suggested that the functional interaction of the caveolin peptide and adenylyl cyclase occurred in a caveolin subtype-dependent manner, as well.



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  		Figure 3. Effect of other caveolin peptides on type V adenylyl cyclase. Type V adenylyl cyclase overexpressed in H5 insect cells was incubated in the absence (-) or presence of 5 µM of various peptides [53–81 on x-axis, the caveolin-1 (Cav 1) nonscaffolding domain peptide, amino acid residues 53–81; 82–101 on x-axis, the caveolin-1 scaffolding domain peptide, amino acid residues 82–101; Cav 2, the caveolin-2 scaffolding domain peptide, amino acid residues 55–73; and Cav 3, the caveolin-3 scaffolding domain peptide, amino acid residues 55–74). To each reaction mixture, 50 µM forskolin was also added. All assays were performed in duplicate, and the results are shown as means ± SEM from four independent experiments. **, P < 0.01, from the values without the peptide.

 
Similar inhibition was observed with type III adenylyl cyclase; type III adenylyl cyclase was inhibited by caveolin-1 (by 34%) and -3 peptide (by 37%) at 5 µM but not by caveolin-2 peptide in forskolin-stimulated condition. We did not examine the effect of caveolin peptide on type III in basal condition because the basal catalytic activity of cells overexpressing type III was marginally higher than that of noninfected cells. In contrast, type II adenylyl cyclase was unaffected by any caveolin peptides in both basal and stimulated conditions (data not shown).

Kinetic analysis
To characterize the mechanism of inhibition, we examined the extent to which the caveolin peptide interfered with the substrate ATP binding. Various concentrations of ATP and of the caveolin-1 scaffolding domain peptide were incubated and subjected to adenylyl cyclase assays. A representative result of the enzyme kinetic studies, in the presence and absence of the peptide, is shown in Fig. 4Go. Lineweaver-Burk analysis showed that the Michaelis constant (Km) value was unaltered, whereas the maximal rate (Vmax) value was remarkably decreased, indicating that the inhibition was not dependent on the competition with the substrate ATP.



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  		Figure 4. Kinetic analysis of the inhibitory effect of the caveolin-1 scaffolding domain peptide. Lineweaver-Burk double-reciprocal plot. Type V adenylyl cyclase overexpressed in H5 insect cells was used. Various concentrations of the substrate ATP were incubated in the presence (Peptide 2.5 µM, closed triangles, with 2.5 µM caveolin peptide; Peptide 5 µM, open triangles, with 5 µM caveolin peptide) or absence (Control, circles) of the caveolin-1 scaffolding domain peptide. Adenylyl cyclase assays were performed in the presence of forskolin (50 µM). The Km value was unchanged in the absence and presence of the peptide (57–59 µM). In contrast, the Vmax value was decreased in the presence of the peptide (3.91 nmol/mg·min in the absence of the peptide; 2.55 nmol/mg·min in the presence of 2.5 µM peptide; and 1.30 nmol/mg·min in the presence of 5.0 µM peptide). A representative result from three independent assays is shown.

 
Effect of mutation of the caveolin-1 peptide
To examine the amino acid sequence requirement for the inhibition more specifically, we tested a series of smaller synthetic peptides derived from the caveolin-1 scaffolding domain: 20-mer (amino acid residues 82–101), 18-mer (amino acid residues 84–101), 16-mer (amino acid residues 86–101), 14-mer (amino acid residues 88–101), and 12-mer (amino acid residues 90–101). As shown in Fig. 5Go, sequential N-terminal deletion of the caveolin-1 peptide decreased its inhibitory effect on type V adenylyl cyclase; the 16-mer peptide retained most of the inhibitory effect, whereas the 12-mer was mostly ineffective. We also examined the effect of C-terminal deletion using a 12-mer peptide (DGIWKASFTTFT, amino acid residues 82–92), in which C-terminal 8-amino acid residues were deleted. In the presence of this peptide, the catalytic activity of type V adenylyl cyclase was unchanged (100.8% of the catalytic activity in the absence of the peptide; mean from two independent determinations), suggesting that the caveolin peptide-mediated inhibition was length-dependent.



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  		Figure 5. Effect of deletion mutagenesis on the caveolin-1 peptide. The caveolin-1 scaffolding domain peptide (82–101 on x-axis, 20-mer, amino acid residues 82–101) was sequentially deleted from the N-terminus, producing 4 deleted peptides (84–101 on x-axis, 18-mer, amino acid residues 84–101; 86–101 on x-axis, 16-mer, amino acid residues 86–101; 88–101 on x-axis, 14-mer, amino acid residues 88–101; and 90–101 on x-axis, 12-mer, amino acid residues 90–101). Type V adenylyl cyclase overexpressed in H5 cells was incubated with 50 µM forskolin in the absence [(-), open bar] and presence of 10 µM each peptide (closed bar, 20-mer; hatched bars, deleted peptides) for assays. Means from two independent experiments are shown.

 
We also assessed the effect of point mutations using the 16-mer peptide used in the above assay. We replaced each residue sequentially with alanine to alter the secondary structure of the caveolin-1 scaffolding domain peptide. As shown in Fig. 6Go, the inhibitory effect was negated, in particular, by the substitution of aromatic residues (Phe89, 92, and 99; Tyr97 and 100; Trp98) and a few nonaromatic residues (Thr93 and Val94) in the middle of the peptide. An N-terminal aromatic residue (Trp85) was mutated also to alanine in the 20-mer peptide (amino acid residue 82–101). In the presence of this 20-mer peptide, the catalytic activity of type V adenylyl cyclase was unchanged (97.6% of the catalytic activity in the absence of the peptide; mean from two independent determinations). Thus, the inhibitory effect was, again, negated by this mutation.



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  		Figure 6. Effect of alanine scanning mutagenesis. Each amino acid residue with the active 16-mer caveolin-1 peptide (amino acid residues 86–101) was point-mutated to alanine. Type V adenylyl cyclase overexpressed in H5 cells was incubated with 50 µM forskolin in the absence (open bar) or presence of 5 µM of each peptide [(+), closed bar, the original 16-mer peptide; hatched bars, mutated peptides]. The amino acid residue mutated to alanine is indicated on the x-axis. Means from two independent experiments are shown.

 
Putting together the inhibitory effect of the caveolin 1 scaffolding domain peptide required the presence of intact aromatic residues and was abolished by shortening the peptide length to 12-mers in either direction (C- vs. N-terminal).

Effect of adenylyl cyclase stimulation
The above experiments were all conducted in the forskolin-stimulated condition of adenylyl cyclase. To test whether the condition of adenylyl cyclase stimulation altered the degree of inhibition by the caveolin-1 scaffolding domain peptide, we compared the peptide effect on type V adenylyl cyclase activity in either stimulated (Gs{alpha} and/or forskolin) or unstimulated (basal) conditions using HEK293 cells stably overexpressing type V adenylyl cyclase. This cell line showed an approximately 5-fold increase in forskolin-stimulated membrane catalytic activity from nontransfected cell line, as we previously reported (26). As shown in Fig. 7Go, regardless of the kind of stimulators, the peptide inhibited adenylyl cyclase dose-dependently. The degree of inhibition, however, was subject to the condition of stimulation and was greater in stimulated conditions (GTP{gamma}S and/or forskolin) than in basal condition.



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  		Figure 7. Effect of stimulating adenylyl cyclase by various stimulators. HEK293 cell membranes stably overexpressing type V adenylyl cyclase were used. The effect of the caveolin-1 scaffolding domain peptide (0–10 µM) was examined in the absence (Basal, open circles) and presence of various stimulators (GTP{gamma}S, closed circles, 100 µM GTP{gamma}S; Fsk, open triangles, 50 µM forskolin; GTP{gamma}S + Fsk, closed triangles, 100 µM GTP{gamma}S and 50 µM forskolin). All assays were done in duplicate and means from two independent experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated that the peptide derived from the caveolin scaffolding domain directly inhibited adenylyl cyclase in an adenylyl cyclase isoform- and a caveolin subtype-dependent manner. The type V and type III isoforms were potently inhibited by the caveolin peptide, whereas type II was resistant to inhibition. The specificity of this inhibition was verified by the peptide mutational analysis and by the inability of the other related peptides (the caveolin-2 peptide and the nonscaffolding domain peptide) to inhibit the same adenylyl cyclase isoform. Mutational analysis of caveolin peptide revealed the importance of the length of the peptide, as well as the presence of intact aromatic residues within the peptide, findings reminiscent of those with G protein (19). Our findings suggest that the role of caveolin in G protein-coupled signaling is more than just accumulating the receptor, G proteins, and adenylyl cyclase in caveolae; its role is also that of regulating their function.

The mechanism of inhibition by the caveolin peptide seems to be different from that by an intrinsic type V adenylyl cyclase peptide, which also inhibits adenylyl cyclase catalytic activity. We previously demonstrated that a 20-mer peptide, derived from the most conserved catalytic domain of type V adenylyl cyclase, inhibited type V adenylyl cyclase by decreasing the Vmax value (22). Although the caveolin peptide inhibited type V adenylyl cyclase similarly by decreasing the Vmax value in kinetic analysis, the intrinsic peptide inhibited the type II adenylyl cyclase isoform, as well. Further, the intrinsic peptide inhibited many cultured cell membrane adenylyl cyclases, indicating that the inhibition of adenylyl cyclase by the intrinsic peptide was not isoform-specific.

Caveolin-mediated inhibition, in our study, and previously demonstrated Gi-mediated inhibition of adenylyl cyclase may be similar in some aspects. In experiments using the insect cell membrane overexpressing the adenylyl cyclase isoforms, both caveolin and Gi inhibited type V adenylyl cyclase but not type II (36, 37). The inhibition by caveolin and by Gi became greater when adenylyl cyclase was in stimulated condition (forskolin and/or Gs{alpha}) than in basal condition. On the other hand, a major difference between the caveolin-mediated inhibition and Gi-mediated inhibition was that caveolin-mediated inhibition of adenylyl cyclase was caveolin subtype-dependent, whereas Gi-mediated inhibition was Gi subtype-independent (37). A peptide derived from caveolin-1 and -3 inhibited adenylyl cyclase, but not caveolin-2. In contrast, all three Gi subtypes (Gi-1, -2, and -3) inhibited adenylyl cyclase with a similar potency (37).

We do not know, however, the extent of each adenylyl cyclase isoform regulation in intact cells by Gi, which is still in controversy (37, 38), and by caveolin. Nevertheless, both Gi and caveolin are highly enriched in caveolae and may both suppress the activity of adenylyl cyclase. Given that the specific activity of certain adenylyl cyclase isoforms is much higher than others, as demonstrated using the isoform-specific expression system and the purified enzymes thereafter (22, 23, 39, 40, 41), cellular mechanisms may exist to avoid excessive accumulation of intracellular cAMP produced by these isoforms, which may include rapid degradation of cAMP by phosphodiesterase, P-site inhibition, tonic inhibition of adenylyl cyclase by Gi may occur isoform-dependently (21, 42, 43, 44). We speculate that caveolin may provide another mechanism to reduce the intracellular cAMP concentration; caveolin may keep adenylyl cyclase quiescent in caveolae.

Most importantly, our findings also suggest that the caveolin peptide may be useful as an adenylyl cyclase isoform-selective inhibitor. Currently, no isoform-selective peptide inhibitors are available with such a high selectivity as the caveolin peptide. Such inhibitors may be valuable in inhibiting cAMP signaling transmitted by a specific isoform(s). It may also lead to the development of drugs that inhibit cAMP signaling transmitted by specific isoforms and, thus, with an improved tissue-selectivity.


   Footnotes
 
1 This study was supported by grants from the United States Public Health Service (HL-38070 and HL-54895), Grant GM-50443 from the United States Public Health Service (to M.P.L.), a grant from Elsa U. Pardee Foundation, a Scholarship in Medical Sciences from the Charles E. Culpeper, and a grant from G. Harold and Leila Y. Mothers Charitable Foundation. Back

Received September 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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