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J. Biol. Chem., Vol. 275, Issue 39, 30211-30219, September 29, 2000

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Heterologous Desensitization Mediated by G Protein-specific Binding to Caveolin^*

Karnam S. Murthy and Gabriel M. Makhlouf

From the Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711

Received for publication, March 13, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We examined the notion that sequestration of G protein subunits by binding to caveolin impedes G protein reassociation and leads to transient, G protein-specific desensitization of response in dispersed smooth muscle cells. Cholecystokinin octapeptide (CCK-8) and substance P (SP) were used to activate G_q/11, cyclopentyl adenosine (CPA) was used to activate G_i3, and acetylcholine (ACh) was used to activate both G_q/11 and G_i3 via m3 and m2 receptors, respectively. CCK-8 and SP increased only G_q/11, and CPA increased only G_i3 in caveolin immunoprecipitates; caveolin and other G proteins were not increased. ACh increased both G_q/11 and G_i3 in a time- and concentration-dependent fashion: only G_q/11 was increased in the presence of an m2 antagonist, and only G_i3 was increased in the presence of an m3 antagonist. To determine whether transient G protein binding to caveolin affected subsequent responses mediated by the same G protein, PLC- activity was measured in cells stimulated sequentially with two different agonists that activate either the same or a different G protein. After treatment of the cells with ACh and an m2 antagonist, the phospholipase C- (PLC-) response to CCK-8 and SP, but not CPA, was decreased; conversely, after treatment of the cells with ACh and an m3 antagonist, the PLC- response to CPA, but not CCK-8 or SP, was decreased. Similarly, after treatment with CCK-8 or SP, the PLC- response mediated by G_q/11 only was decreased, whereas after treatment with CPA, the PLC- response mediated by G_i3 only was decreased. A caveolin-binding G_q/11 fragment blocked the binding of activated G_q/11 but not G_i3 to caveolin-3 and prevented desensitization of the PLC- response mediated only by other G_q/11-coupled receptors. A caveolin-binding G_i3 fragment had the reverse effect. Thus, transient binding of receptor-activated G protein subunits to caveolin impedes reassociation of the heterotrimeric species and leads to desensitization of response mediated by other receptors coupled to the same G protein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular cloning has identified three distinct genes encoding caveolins, the main structural proteins of caveolae (1-7). Caveolins (caveolin-1 and caveolin-1, caveolin-2, and caveolin-3) exhibit tissue-specific distribution: caveolin-1 and caveolin-2 are co-localized and abundantly expressed in adipocytes, endothelial cells, and fibroblasts; caveolin-3 is expressed exclusively in skeletal, cardiac and smooth muscle and is not co-localized with caveolin-2 except in undifferentiated cells (3, 8, 9). Caveolin-1 and caveolin-3 form large homo-oligomers (200-440 kDa); in addition, caveolin-1 associates with caveolin-2 to form larger hetero-oligomers (8, 10, 11).

The structure of caveolin monomers is similar, with a central 33-amino acid segment and hydrophilic N-terminal (70-101 amino acids) and C-terminal (43-44 amino acids) segments facing the cytoplasm (3, 7, 12). The C-terminal segment directs the formation of homo-oligomers. A short cytosolic domain within this segment (residues 82-101 in caveolin-1, 54-73 in caveolin-2, and 55-74 in caveolin-3) termed the "caveolin-scaffolding domain" determines the binding of caveolin to various signaling molecules, such as G protein subunits (13-15), endothelial nitric-oxide synthase (16, 17), protein kinase C isoforms (18, 19), G protein-coupled receptor kinases (20), and a variety of receptor and cytosolic tyrosine kinases (Ras and Src family tyrosine kinases, mitogen-activated protein kinases, and epidermal and platelet-derived growth factor receptors (21-27)). Upon activation, some G protein-coupled receptors (e.g. endothelin-A (28), bradykinin-B₂ (29), ₂ adrenergic (30), and muscarinic m2 receptors (31)) translocate to caveolae where they bind to caveolin.

Compelling evidence exists that a variety of signaling events are initiated in caveolae (16, 22, 42), consistent with the notion that caveolin provides a scaffold for the assembly of signaling molecules into modules primed for activation. Caveolin could also act to restrain cellular response by selective binding of signaling molecules, such as G proteins. Caveolin-binding motifs consisting of 10-15-mer sequences with characteristically spaced aromatic residues (XXXXX or XXXXXX, where  is the aromatic amino acid Trp, Phe, or Tyr), are present in all caveolin-binding proteins, including G proteins (13). Binding of G proteins to caveolin could lead to their sequestration and enrichment in inactive form in caveolar microdomains (6). Pharmacological activation with GTPS¹ strongly inhibits and mutational activation abolishes G protein binding to caveolin consistent with preferential interaction of caveolin with GDP-bound G subunits (15).

The possibility that receptor-activated G protein subunits are sequestered by binding to caveolin or a caveolin-associated protein, leading to transient, G protein-specific desensitization of response, has been raised but not experimentally tested (29). This notion was examined in the present study using a series of agonists previously shown to activate G_q/11 (cholecystokinin octapeptide, and substance P), G_i3 (cyclopentyl adenosine), or both G_q/11 and G_i3 (acetylcholine) in smooth muscle (33-37). Caveolin-binding fragments of G_q/11 and G_i3 were used to inhibit competitively G binding to caveolin and suppress desensitization of response (13, 15). The results show that receptor activation was followed by transient binding of activated G to caveolin that was selectively blocked by caveolin-binding fragments. Binding of G to caveolin resulted in transient desensitization of cellular response mediated by other receptors coupled to the same G protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Dispersed Smooth Muscle Cells-- Muscle cells were isolated from the circular muscle layer of rabbit intestine by enzymatic digestion at 31 °C with collagenase, followed by filtration though 500-µm Nitex mesh and low speed centrifugation as described previously (33, 35, 37). The cells were suspended in HEPES medium containing 120 mM NaCl, 2.5 mM KH₂PO₄, 4.0 mM KCl, 0.6 mM MgCl₂, 25 mM HEPES, and 2.1% essential amino acid mixture. In some experiments, muscle cells were permeabilized by 5-min treatment with saponin (35 µg/ml) and resuspended in low Ca²⁺ (100 nM) medium (37).

Identification of Receptor-activated G Proteins-- G proteins selectively activated by various receptors were identified by the method of Okamoto et al. and others (37, 38). Muscle cell homogenates were centrifuged at 27,000 × g for 15 min, and the crude membranes were suspended in 20 mM HEPES medium (pH 7.4) containing 2 mM EDTA and 240 mM NaCl. The membranes were diluted 20-fold and incubated at 37 °C with 60 nM [³⁵S]GTPS in a medium containing 10 mM HEPES (pH 7.4), 100 µM EDTA, and 10 mM MgCl₂. The reaction was stopped with 10 volumes of 100 mM Tris-HCl medium (pH 8.0) containing 10 mM MgCl₂, 100 mM NaCl, and 20 µM GTP, and the mixture was incubated for 2 h on ice in wells coated with specific G protein antibodies. The wells were washed with phosphate buffer solution containing 0.05% Tween 20, and the radioactivity from each well was counted.

Assay for PLC- Activity-- PLC- activity was measured from the formation of total inositol phosphates in muscle cells prelabeled with myo-[³H]inositol as described previously (39). 10 ml of cell suspension (2 × 10⁶ cell/ml) were labeled with myo-[³H]inositol (15 µCi/ml) for 3 h at 31 °C. The cells were centrifuged at 350 × g for 10 min and resuspended in 10 ml of fresh HEPES medium. The cells were treated with one agonist for 10 min and centrifuged again at 350 × g for 5 min. Various agonists were then added to 0.5 ml of cell suspension for 30 s, and the reaction was terminated with 940 µl of chloroform:methanol:HCl (50:100:1 v/v/v). After chloroform (310 µl) and water (310 µl) were added, the samples were vortexed, and the phases were separated by centrifugation at 1000 × g for 15 min. The upper aqueous phase was applied to Dowex AG-1 × 8 columns. After washing, inositol phosphates were eluted with 5 ml of 0.8 M ammonium formate with 0.1 M formic acid, and the eluates were collected into scintillation vials and counted in gel phase after addition of 10 ml of scintillant. The results were expressed as cpm/10⁶ cells.

Caveolin Immunoprecipitation and Immunoblotting of G Proteins-- Smooth muscle cells (2-3 × 10⁶ cells/ml) were lysed by incubation for 30 min at 4 °C in 10 mM Tris (pH 7.5), 50 mM NaCl, 1% Triton X-100, and 60 mM octyl glucoside, and the lysate was centrifuged at 15,000 × g for 30 min. The supernatant was precleared by incubation with 0.1% albumin-coated protein A-Sepharose for 6 h at 4 °C and then incubated overnight with polyclonal caveolin-3 antibody at a final concentration of 4 µg/ml. Protein A-Sepharose was then added for 1 h, and the mixture was centrifuged for 5 min. The immunoprecipitates were washed four times with lysis buffer and boiled in Laemmli buffer. Samples were separated by SDS-PAGE in 12% acrylamide gel, electrotransferred to nitrocellulose paper, and probed with antibodies to G_i3, G_q/11, or caveolin-3. After incubation with secondary antibody conjugated with horseradish peroxidase, the proteins were visualized using the Super Signal ULTRA chemiluminescent substrate. The intensity of the protein band on Hyperfilm-ECL was determined by densitometry.

Detergent-free Purification of Caveolin-enriched Membrane Fractions-- Caveolin-enriched membrane fractions derived from intestinal smooth muscle were prepared by the method of Song et al. (9). Dispersed muscle cells were washed three times in phosphate-buffered saline and suspended in 2 ml of 500 mM sodium carbonate (pH 11.0) containing 0.2 mM of phenylmethylsulfonyl fluoride and 20 µg/ml of leupeptin and homogenized with a Polytron tissue grinder (three 10-s bursts) and by sonication (three 20-s bursts). The homogenate was adjusted to 45% sucrose in MBS (25 mM Mes, pH 6.5, and 0.15 M NaCl), placed in an ultracentrifuge tube and overlaid with two 4-ml layers of 35 and 5% sucrose in MBS containing 250 mM sodium carbonate. The gradient was centrifuged at 39,000 rpm for 20 h. Twelve 1-ml fractions were collected sequentially from the top and designated as fractions 1-12. Fractions were analyzed by SDS-PAGE (15% acrylamide gels); after transfer to nitrocellulose membranes, Western blot analysis was performed with antibodies to caveolin-3 and various G subunits. Immunoreactive bands were visualized by 1-h incubation with horseradish peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence assay.

For protein immunoprecipitation from caveolin-enriched fractions, the purified membranes were diluted to 2 mg protein/ml in lysis buffer, incubated on ice for 1 h, and centrifuged. The lysate was precleared by 1-h incubation with protein A-Sepharose and then incubated overnight with caveolin-3 antibody and for 2 h with protein A-Sepharose. Immunoprecipitates were washed five times with lysis buffer and resuspended in 30 µl of 2-fold concentrated Laemmli buffer; after separation on SDS-PAGE and transfer to nitrocellulose membranes, immunoblot analysis with caveolin-3 and G antibodies was performed.

Phosphatidylinositol 4,5-Bisphosphate Assay in Caveolar Membranes-- PIP₂ was measured by thin layer chromatography in caveolar membranes as described previously (40, 41). A 20-ml cell suspension (10⁶ cells/ml) was incubated with 500 µCi of [³²P]P_i at 31 °C for 3 h. Duplicate samples (10⁶ cells/ml) were incubated at 31 °C with ACh (0.1 µM), cholecystokinin-8 (CCK-8 1 nM), SP (1 µM), and CPA (1 µM) separately for 30 s. The reaction was terminated by centrifugation at 15,000 × g for 5 min followed by addition of 1 ml of HEPES buffer (25 mM) containing 0.5% Triton X-100. The mixture was incubated for 10 min and then centrifuged at 15,000 × g for 5 min. Supernatant and pellets were extracted with 1.8 ml of chloroform-methanol-HCl (100:200:2 v/v/v). The organic phase was analyzed for PIP₂ by thin layer chromatography. The results were expressed as cpm/10⁶ cells.

[³H]Scopolamine Binding to Smooth Muscle Cells-- Binding of [³H]scopolamine to dispersed intestinal smooth muscle cells was done as described previously (37). Muscle cells were suspended in HEPES medium containing 1% bovine serum albumin. Triplicate 0.5-ml aliquots (10⁶ cells/ml) were incubated for 15 min with 1 nM [³H]scopolamine alone or with acetylcholine. Bound and free radioligand were separated by rapid filtration under reduced pressure through 5-µm polycarbonate Nucleopore filters and washed four times with 3 ml of ice-cold HEPES medium containing 0.2% bovine serum albumin. Nonspecific binding (28 ± 6%) was calculated as the amount of radioactivity in the presence of 10 µM acetylcholine. [³H]Scopolamine binding was measured in control cells and in cells treated for 10 min with 1 nM CCK-8, 1 µM SP, 1 µM CPA, or 0.1 µM ACh.

Materials-- Two peptides corresponding to the caveolin-binding domains of G_q/11, Tyr¹⁹²-Ala²⁰⁶ (YPFDLQSVIFRMVDA) and G_i3, Thr¹⁸⁷-Val²⁰¹ (THFTFKELYFKMFDV) were synthesized by the solid phase method and purified (95-99%) by high performance liquid chromatography (Peptidogenic, CA). [2-³H]Inositol, [³H]scopolamine, [³⁵S]GTPS, and [³²P]orthophosphate were obtained from NEN Life Science Products; Dowex AG-1 × 8 resin was from Bio-Rad; polyclonal G_q/11, G_i1/2, and G_i3 antibodies were from Santa Cruz Biotechnology; and caveolin-3 antibody was from Transduction Laboratories (Lexington, KY). All other reagents were from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Agonist-induced Activation of G Proteins-- G protein activation was determined directly from the increase in agonist-stimulated binding of [³⁵S]GTPS to specific G subunits in solubilized intestinal smooth muscle cell membranes. CCK-8 and SP selectively activated G_q/11, whereas CPA selectively activated G_i3 (Table I). ACh activated both G_q/11 and G_i3 via m3 and m2 receptors, respectively, as shown previously (37).

Distribution of Caveolin-3 and G Proteins in Caveolar Membranes in the Basal State and after Stimulation with Agonists-- Immunoblot analysis of 12 fractions derived from intestinal smooth muscle cells showed that caveolin-3 was confined to low density fractions 5 and 6, whereas G proteins (G_q, G_i1/2, and G_i3) were present in fractions 5 and 6, as well as in membrane fractions 9-12 (Fig. 1). The pattern was similar to that obtained in other cell types (15, 29).

View larger version (68K): [in this window] [in a new window]   Fig. 1.   Location of caveolin-3 and G subunits in smooth muscle membrane fractions. Twelve sucrose-density gradient fractions were prepared as described under "Experimental Procedures" and subjected to SDS-PAGE and Western blot analysis with specific antibodies to caveolin-3 and G subunits. Caveolin-3 was confined to low density fractions 5 and 6. G subunits were present in fractions 5 and 6, as well as fractions 9-12.

A specific redistribution of G proteins to caveolin-enriched fractions (combined fractions 5 and 6) occurred upon treatment of smooth muscle cells with agonists. Treatment with CCK-8 (1 nM) for 10 min caused a significant increase of G_q/11 (154 ± 18%; p < 0.01) but not G_i3, G_i2, or G_i1 in caveolin immunoprecipitates (Fig. 2). Conversely, treatment with CPA (1 µM) caused a significant increase of G_i3 (168 ± 24%; p < 0.01), but not G_q/11, G_i2, or G_i1, in caveolin immunoprecipitates (Fig. 2). There was no change in caveolin-3 after treatment with either agonist (Fig. 2). The increase in G binding to caveolin paralleled agonist-induced activation of the corresponding G protein (Table I).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Immunoblot analysis of G protein subunits and caveolin-3 in caveolin immunoprecipitates. Caveolin immunoprecipitates were obtained from caveolin-enriched fractions (fractions 5 and 6 in Fig. 1) derived from muscle cells treated for 10 min with CCK-8 (1 nM) or CPA (1 µM). Immunoblot analysis with G and caveolin-3 antibodies was then performed. A selective increase of Gq/11 with CCK-8 (p < 0.01), and of Gi3 with CPA (p < 0.01) was observed, without increase in Gi1/2 or caveolin-3. Results are expressed as percentages of basal level. Values are the means ± S.E. of three experiments.

View this table: [in this window] [in a new window]   Table I Selective activation of G proteins by agonists CHAPS-solubilized smooth muscle membranes were incubated for 20 min with 60 nM [35S]GTPS alone or with various agonists and then added to wells coated with specific G antibodies (Ab). Values are the means ± S.E. of three experiments.

Distribution of G Proteins in Caveolin Immunoprecipitates after Stimulation with Agonists-- A similar pattern was obtained in caveolin immunoprecipitates derived from cell lysates. Treatment of muscle cells with CCK-8 or SP increased G_q/11 in caveolin-3 immunoprecipitates by 187 ± 22% (p < 0.01) and 181 ± 24% (p < 0.01), respectively, but had no effect on G_i3 (Fig. 3). In contrast, treatment of muscle cells with CPA (1 µM) increased G_i3 in caveolin-3 immunoprecipitates by 173 ± 18% (p < 0.01) but had no effect on G_q/11 (Fig. 3). G_i2 and G_i1 are not activated by CCK-8, SP, or CPA (Table I) and did not increase in caveolin-3 immunoprecipitates upon treatment of muscle cells with all three agonists (Fig. 4). There was no increase in caveolin-3 in caveolin immunoprecipitates or in G_q/11 and G_i3 immunoprecipitates after treatment with any agonist (Fig. 4).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Pattern of increase of Gq/11 and Gi3 in caveolin-3 immunoprecipitates induced by different agonists. Dispersed smooth muscle were treated for 10 min with various agonists (1 nM CCK-8, 1 µM SP, 1 µM CPA, and 0.1 µM ACh alone or with 4-DAMP (m3 antagonist) and methoctramine (meth, m2 antagonist). Increase of Gq/11 (upper panel) and Gi3 (lower panel) in caveolin-3 immunoprecipitates was determined by immunoblot analysis. Values are the means ± S.E. of four experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Immunoblot analysis of Gi1/2 and caveolin-3. Whole cell lysates were subjected to immunoprecipitation with caveolin-3 antibody (A) and Gi3 or Gq/11 antibody (B). Samples from caveolin immunoprecipitates were immunoblotted with Gi1/2 or caveolin-3 antibody; samples from Gq/11 and Gi3 immunoprecipitates were immunoblotted with caveolin-3 antibody. There was no increase in caveolin-3 or Gi1/2 from basal levels after treatment with various agonists. The m2 but not the m3 receptor was bound to caveolin-3 after treatment with ACh (C).

The pattern of selective increase in G subunits was reinforced by analysis of caveolin-3 immunoprecipitates after stimulation with ACh. Treatment of muscle cells with ACh (0.1 µM) increased both G_q/11 and G_i3 in caveolin-3 immunoprecipitates by 177 ± 29% (p < 0.01) and 181 ± 37% (p < 0.01), respectively (Fig. 3). The increase in G_q/11 was abolished by the m3 receptor antagonist, 4-DAMP, but was not affected by the m2 receptor antagonist, methoctramine, whereas the increase in G_i3 was abolished by methoctramine but was not affected by 4-DAMP (Fig. 3). In these experiments also there was no increase in G_i1 or G_i2 in caveolin immunoprecipitates and no increase in caveolin-3 either in caveolin immunoprecipitates or G_q/11 and G_i3 immunoprecipitates (Fig. 4). The m2 but not m3 receptors were detected in caveolin immunoprecipitates after treatment of muscle cells with ACh (Fig. 4C).

Stimulation with agonists that activate G_i1 and/or G_i2 showed that caveolin binding was not confined to G_q/11 or G_i3. Treatment of muscle cells with cANP4-23 (1 µM), a selective ligand of the natriuretic peptide clearance receptor, NPR-C, shown recently to couple to both G_i1 and G_i2 (42), increased G_i1 by 199 ± 23% (p < 0.01) and G_i2 by 212 ± 20% (p < 0.01) in caveolin-3 immunoprecipitates. Treatment with somatostatin-14 (1 µM), which activates G_i1 in smooth muscle (43), increased G_i1 by 192 ± 20% (p < 0.01) in caveolin-3 immunoprecipitates, whereas treatment with [D- Pen^2,5]enkephalin, which activates G_i2 (44), increased Gi₂ by 190 ± 18% (p < 0.01). In contrast to agonists, activation of G proteins with GTPS (100 µM) in permeabilized smooth muscle cells did not increase G_q/11 (11 ± 17%; NS) or G_i3 (8 ± 14%; NS) in caveolin-3 immunoprecipitates consistent with preferential binding of caveolin to inactive GDP-bound G subunits (15).

A common  antibody was used to determine whether subunits increased in caveolin-3 immunoprecipitates. Both CCK-8 and CPA increased G in caveolin-3 immunoprecipitates by 182 ± 21% (p < 0.01) and 232 ± 28 (p < 0.01), respectively.

Time Course and Concentration Dependence of Agonist-stimulated Increase of G in Caveolin-3 Immunoprecipitates-- Treatment of smooth muscle cells with a maximal concentration of ACh (0.1 µM) caused a time-dependent increase of G_q/11 and G_i3 in caveolin-3 immunoprecipitates that attained a peak in 5 min. The peak was sustained for 15 min and declined rapidly to control levels in the next 20 min (Fig. 5). The peak increase was concentration-dependent (Fig. 6).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Time course of acetylcholine-stimulated binding of Gq/11 and Gi3 to caveolin-3 in smooth muscle. Dispersed smooth muscle were treated with ACh (0.1 µM) for various periods. After lysis, caveolin-3 immunoprecipitates were subjected to SDS-PAGE and probed with Gq/11 and Gi3 antibodies; the bands were analyzed by densitometry. Values are the means ± S.E. of four experiments. p < 0.01 for binding between 2 and 30 min.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent stimulation of Gq/11 and Gi3 binding to caveolin-3 by acetylcholine. Dispersed smooth muscle were treated with various concentrations of ACh for 10 min, and the binding of Gq/11 and Gi3 to caveolin-3 was determined. Values are the means ± S.E. of 3-7 experiments. p < 0.01 at all concentrations.

Inhibition of G Protein Binding to Caveolin-3 by Caveolin-binding G Protein Fragments-- G protein fragments that selectively bind to the caveolin-scaffolding domain were used to block the binding of activated G proteins to caveolin-3. Addition of the caveolin-binding G_q/11 fragment, YPFDLQSVIFRMVDA (50 µM), to permeabilized muscle cells for 10 min blocked the increase in G_q/11 binding to caveolin elicited by SP but not the increase in G_i3 binding elicited by CPA (Fig. 7). Conversely, addition of the G_i3 fragment, THFTFKELYFKMFDV (50 µM), blocked the increase in G_i3 binding to caveolin-3 elicited by CPA but not the increase in G_q/11 binding elicited by SP (Fig. 7).

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Inhibition of G protein binding to caveolin-3 by caveolin-binding G protein fragments. Permeabilized smooth muscle were incubated with for 10 min with SP (1 µM) or CPA (1 µM) alone and in combination with Gq/11 or Gi3 fragments that selectively bind to the caveolin-scaffolding domain. Caveolin-3 immunoprecipitates were probed with Gq/11 and Gi3 antibodies, and the bands were analyzed by densitometry. The caveolin-binding Gq/11 fragment blocked the increase in Gq/11 binding to caveolin elicited by SP but not the increase in Gi3 binding elicited by CPA. Conversely, the Gi3 fragment blocked the increase in Gi3 binding to caveolin-3 elicited by CPA but not the increase in Gq/11 binding elicited by SP. Bands denote representative experiments. Values denoted by bars are the means ± S.E. of three experiments.

Heterologous Desensitization of PLC- Activity Mediated by G Protein-specific Binding to Caveolin-- The possibility that binding of activated G proteins to caveolin-3 could result in desensitization of response was tested by sequential stimulation of muscle cells with different agonists that couple to the same or a different G protein. PLC- activity in response to CCK-8 or CPA was measured in muscle cells pretreated for periods ranging from 5 to 60 min with 0.1 µM ACh or for 10 min with different concentrations of ACh (10 pM to 0.1 µM). PLC- activity in response to both CCK-8 and CPA decreased in parallel with the time of pretreatment with ACh, attaining a maximum in cells pretreated for 5-10 min and reverting to control levels in cells pretreated for 40 min (Fig. 8). The decrease in PLC- activity paralleled the increase in G binding to caveolin (Figs. 5 and 8). Pretreatment with different concentrations of ACh for 10 min decreased the PLC- response to CCK-8 and CPA in a concentration-dependent fashion (Fig. 9). In both concentration response and time course studies, there was a close linear correlation (r = 0.99) between the decrease in PLC- activity and the increase in caveolin-bound G_q/11 or G_i3 (Fig. 10).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Time course of inhibition of CCK- and CPA-stimulated PLC- activity after treatment of muscle cells with ACh. Dispersed smooth muscle cells were labeled with [3H]myo-inositol and treated for different periods with ACh (0.1 µM). After washing, the cells were restimulated with either CCK-8 (1 nM) or CPA (1 µM). PLC- activity was determined from the formation of [3H]inositol phosphates expressed in cpm/106 cells above basal levels (406 ± 42 cpm/106 cells). Values are the means ± S.E. of four experiments. p < 0.01 for all values between 5 and 30 min.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Inhibition of CCK- and CPA-stimulated PLC- activity after treatment with various concentrations of ACh. Dispersed smooth muscle cells were labeled with [3H]myo-inositol and treated for 10 min with various concentrations of ACh. After washing, the cells were restimulated with either CCK-8 (1 nM) or CPA (1 µM). PLC- activity was determined from the formation of [3H]inositol phosphates expressed in cpm/106 cells above basal levels (387 ± 44 cpm/106 cells). Values are the means ± S.E. of four experiments. *, p < 0.05; **, p < 0.01 decrease in PLC- activity from control.

View larger version (21K): [in this window] [in a new window]   Fig. 10.   Correlation between binding of G subunits to caveolin-3 and inhibition of PLC- activity. Data from Figs. 5, 6, 8, and 9 were used to determine the correlation between the increase in caveolin-bound Gq/11 or Gi3 induced by ACh and the inhibition of CCK- and CPA-stimulated PLC- activity after treatment with ACh (linear correlations, r = 0.99 for each slope).

Pretreatment with ACh after blockade of m3 receptors with 4-DAMP decreased PLC- activity in response to CPA (71 ± 3%) but not CCK-8 (9 ± 6%) or SP (7 ± 5%) (Fig. 11), whereas pretreatment with ACh after blockade of m2 receptors with methoctramine decreased PLC- activity in response to CCK-8 (51 ± 6%) and SP (49 ± 3%) but not CPA (Fig. 11). Pretreatment with ACh after blockade of both m3 and m2 receptors did not decrease PLC- activity in response to any agonist, including ACh itself (range of decrease, 1 ± 7 to 10 ± 5%).

View larger version (32K): [in this window] [in a new window]   Fig. 11.   Inhibition of CCK-, SP-, and CPA-stimulated PLC- activity after treatment of muscle cells with ACh alone or with specific m2 and m3 antagonists. Dispersed smooth muscle cells were labeled with [3H]myo-inositol and treated for 10 min with ACh (0.1 µM) alone or with 4-DAMP (m3 antagonist) and methoctramine (m2 antagonist). After washing, the cells were restimulated with either CCK-8 (1 nM), SP (1 µM), or CPA (1 µM). PLC- activity was determined from the formation of [3H]inositol phosphates expressed in cpm/106 cells above basal levels (412 ± 35 cpm/106 cells). Values are the means ± S.E. of four experiments. **, p < 0.01 decrease in PLC- activity from control.

Desensitization of PLC- Activity after Treatment with Noncholinergic Agonists-- Pretreatment with other agonists besides ACh provided further support for the notion that inhibition of PLC- activity was G protein-specific. Pretreatment with CCK-8 decreased PLC- activity in response to SP (41 ± 4%) and ACh (56 ± 4%) but not CPA (Fig. 12). Similarly, pretreatment with SP decreased PLC- activity in response to CCK-8 (52 ± 4%) and ACh (51 ± 8%) but not CPA. In contrast, pretreatment with CPA decreased PLC- activity in response to ACh (26 ± 3%) but not CCK-8 or SP (Fig. 12). Thus, pretreatment with one agonist inhibited the response to a different agonist that activated the same G protein.

View larger version (29K): [in this window] [in a new window]   Fig. 12.   Inhibition of PLC- activity stimulated by various agonists after treatment with CCK-8, SP, and CPA. Dispersed smooth muscle cells were labeled with [3H]myo-inositol and treated for 10 min with 1 nM CCK-8, 1 µM SP, or 1 µM CPA. After washing, the cells were restimulated with either CCK-8 (1 nM), SP (1 µM), CPA (1 µM), or ACh (0.1 µM). PLC- activity was determined from the formation of [3H]inositol phosphates expressed in cpm/106 cells above basal levels (388 ± 39 cpm/106 cells). Values are the means ± S.E. of four experiments. *, p < 0.05; **, p < 0.01 decrease in PLC- activity from control.

Finally, pretreatment of muscle cells for 10 min with CCK-8, SP, ACh, or CPA decreased PLC- activity in response to subsequent treatment with the same agonist by 83 ± 3 to 91 ± 4%. The large decrease in PLC- activity reflected homologous desensitization of the receptor as well as desensitization resulting from transient G protein sequestration in caveolae.

Linkage of G Protein-specific Desensitization to G Protein-specific Binding to Caveolin-- Pretreatment of permeabilized muscle cells with ACh (0.1 µM) for 10 min decreased PLC- activity in response to SP (59 ± 5%) or CPA (61 ± 6%) to the same extent as in intact cells. Pretreatment of the cells with ACh and the caveolin-binding G_q/11 fragment (50 µM) blocked the decrease in PLC- activity in response to SP, but not in response to CPA (Fig. 13). Conversely, pretreatment of the cells with ACh and the caveolin-binding G_i3 fragment (50 µM) blocked the decrease in PLC- activity in response to CPA but not in response to SP (Fig. 13). Pretreatment of permeabilized muscle cells with either G protein fragment alone had no effect on control PLC- activity stimulated by SP or CPA (Fig. 13).

View larger version (31K): [in this window] [in a new window]   Fig. 13.   Blockade of desensitization of PLC- response by caveolin-binding Gq/11 and Gi3 fragments. PLC- activity in response to SP or CPA was measured before (solid bars) or after 10-min treatment with ACh (0.1 µM) alone and in combination with Gq/11 or Gi3 fragments (50 µM) (hatched bars). Pretreatment with Gq/11 or Gi3 fragments prevented the decrease in PLC- response to SP and CPA, respectively. Data are the means ± S.E. of three experiments. **, p < 0.01 from control response.

PKC-independent Desensitization of PLC- Activity Mediated by G_q/11 and G_i3-- To rule out the involvement of PKC in heterologous desensitization of PLC- activity, muscle cells were pretreated for 10 min with ACh (0.1 µM) in the presence or absence of calphostin C (1 µM). Pretreatment with ACh in the presence of calphostin C had no effect on the decrease in PLC- activity in response to CCK-8, SP, or CPA, implying that desensitization was not caused by PKC-dependent phosphorylation of G proteins or other protein targets (receptors or effector enzymes) in the PI signaling pathway mediated by G_q/11 or G_i3 in smooth muscle (Fig. 14).

View larger version (25K): [in this window] [in a new window]   Fig. 14.   Lack of effect of PKC inhibition on desensitization of PLC- response. Dispersed smooth muscle cells labeled with [3H]myo-inositol were treated for 10 min with ACh (0.1 µM) alone or in combination with 1 µM calphostin C. After washing the cells were restimulated with CCK-8 (1 nM), SP (1 µM), or CPA (1 µM). PLC- activity was expressed as [3H]inositol phosphates (cpm/106 cells) above basal level (456 ± 54 cpm/106 cells). The decrease in PLC- activity induced by CCK-8, SP, and CPA was not altered by calphostin C. Values are the means ± S.E. of four experiments. **, p < 0.01 for difference from control.

To determine whether pretreatment with various agonists affected receptor binding, [³H]scopolamine binding was measured in dispersed smooth muscle cells before and after treatment with CCK-8, SP, or CPA. Treatment with all three agonists had no effect on [³H]scopolamine binding, whereas treatment with ACh caused a significant decrease in binding, reflecting homologous desensitization of m2 and m3 receptors (Fig. 15).

View larger version (36K): [in this window] [in a new window]   Fig. 15.   [3H]Scopolamine binding to dispersed muscle cells before and after treatment with agonists. Specific [3H]scopolamine binding was measured before and after treatment of muscle cells for 10 min with CCK-8 (1 nM), SP (1 µM), CPA (1 µM), or ACh (0.1 µM). Inhibition of binding was observed after treatment with ACh only. Values are the means ± S.E. of four experiments.

Activation of PLC- in Caveolar Membranes-- To determine whether activation of PLC- occurred in caveolar membranes, PIP₂ levels were measured in both Triton-soluble and -insoluble (i.e. caveolar) fractions before and after treatment with all four agonists. No significant change was observed in PIP₂ levels in the Triton-soluble fraction after treatment with various agonists (control, 2781 ± 203 cpm/10⁶ cells; range with various agonists, 2633 ± 108 to 2729 ± 169 cpm/10⁶ cells). However, each agonist separately decreased PIP₂ levels in Triton-insoluble fractions (ACh, 48 ± 3%; CCK-8, 39 ± 5%; SP, 37 ± 3%; CPA, 34 ± 2%), implying that PIP₂ hydrolysis occurred in caveolae (Fig. 16).

View larger version (31K): [in this window] [in a new window]   Fig. 16.   Agonist-stimulated PIP2 hydrolysis in caveolar membranes. Dispersed smooth muscle cells were labeled with [32P]Pi and stimulated with various agonists (0.1 µM ACh, 1 nM CCK-8, 1 µM SP, or 1 µM CPA). PIP2 levels were measured by thin layer chromatography in Triton-soluble and -insoluble (caveolar) fractions and expressed as cpm/106 cells. Each agonist separately decreased PIP2 levels in Triton-insoluble caveolar fractions. No change in PIP2 levels was found in Triton-soluble fractions. Values are the means ± S.E. of four experiments. **, p < 0.01 decrease in PIP2 levels from control. Inset: Immunoblot analysis of caveolin-3 using equal amounts of protein from Triton-soluble (S) and -insoluble (I) fractions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Receptor desensitization by GRKs and -arrestins and/or by feedback phosphorylation via second messenger-activated protein kinases, chiefly cAMP-dependent protein kinase and protein kinase C (PKC), are well established mechanisms of desensitization of response mediated by G protein-coupled receptors (45). Phosphorylation by second messenger-activated protein kinases does not require receptor occupancy and can thus target both homologous and heterologous receptors, reducing their ability to transduce signals and, in some instances, switching the specificity of receptor coupling to G proteins. Phosphorylation of downstream targets in the signaling pathway (e.g. G proteins or effector enzymes) can also result in desensitization of response (46-51). PKC-dependent phosphorylation, however, is G protein-specific: G_z, G₁₂, and G₁₃ are readily phosphorylated, whereas G_q/11 and G_s are not (47-49). PKC-dependent phosphorylation of pertussis toxin-sensitive G proteins in intestinal smooth muscle was observed with G_i1 and G_i2, but not G_i3 or G_o, and resulted in PKC-dependent desensitization of responses mediated by G_i1 and G_i2 (51). Consistent with these results, a selective PKC inhibitor had no effect on desensitization of responses mediated by G_i3 or G_q/11 in the present study, making it possible to explore other G protein-dependent mechanisms of desensitization. The results indicate that transient sequestration of receptor-activated G protein subunits by binding to caveolin leads to heterologous desensitization of responses mediated by other receptors coupled to the same G protein.

Compelling evidence exists that caveolin can act as a scaffold for the assembly and activation of signaling molecules (16, 22, 32). The present study suggests a novel, complementary function whereby caveolin acts to restrain cellular response by transient, selective binding of G proteins. Receptor-activated G protein subunits interact with an assembly of signaling molecules consisting of an effector enzyme (e.g. PLC-) and its substrate (e.g. PIP₂) located in caveolae. Upon GTP hydrolysis, G.GDP binds with high affinity to caveolin-3 and is transiently sequestered, impeding reassociation of the heterotrimeric G protein. The decrease in the levels of G protein available to receptors in the extracaveolar membrane causes a transient decrease in the ability of agonists to activate PLC-, i.e. a G protein-specific heterologous desensitization of response.

Caveolin Binding of Activated G Proteins-- The increase of G in caveolin immunoprecipitates derived from caveolin-enriched fractions and whole cell lysates was confined to activated G proteins. The G subunits appeared to bind directly to caveolin-3 rather than to a caveolin-associated protein and could be competed out by the corresponding caveolin-binding G protein fragment. Thus, a caveolin-binding G_q/11 fragment blocked the binding of activated G_q/11 but not G_i3 to caveolin-3, whereas a caveolin-binding G_i3 fragment had the reverse effect, blocking the binding of activated G_i3 but not G_q/11 to caveolin-3.

The increase in G_q/11 and G_i3 binding to caveolin-3 induced by ACh was both time- and concentration-dependent, reflecting concurrent activation of m3 receptors coupled to G_q/11 and m2 receptors coupled to G_i3, a notion confirmed by the increase in the binding of only one G protein subunit in the presence of selective m3 or m2 receptor antagonists. The increase induced by other agonists was also G protein-specific, with CCK-8 and SP increasing G_q/11 and CPA increasing G_i3 binding to caveolin. de Weerd and Leeb-Lundberg (29) have shown that activation of bradykinin B₂ receptors that couple to G_q/11 and G_i in DDT₁ MF-2 smooth muscle cells increased G_q and G_i binding to caveolin with a time course similar to that elicited by ACh in intestinal smooth muscle cells. Some receptors, for example, m2 receptors in cardiac myocytes (31) and CCK-A receptors in pancreatic acinar cells (52) also bind to caveolin upon activation. In the present study, m2 but not m3 receptors bound to caveolin-3 upon activation with ACh, implying that translocation of receptors to caveolae was not a prerequisite for desensitization of response, which was observed upon selective activation of either m2 or m3 receptors.

The decrease in caveolar PIP₂ levels suggested that activation of PLC- by G proteins occurred in caveolae. A similar decrease in caveolar PIP₂ levels has been reported in A431 cells stimulated with bradykinin and epidermal growth factor (41, 53, 54). Our previous studies had shown that PIP₂ hydrolysis induced by receptors (e.g. CCK-A and m3 receptors) coupled to G_q/11 was mediated by G-dependent activation of PLC-1, whereas PIP₂ hydrolysis induced by receptors coupled to G_i/o (e.g. somatostatin-3, -opioid, adenosine A1, and muscarinic m2 receptors) was mediated by -dependent activation of PLC-3 (33, 36, 42, 43).

G subunits activated by the nonhydrolyzable analog, GTPS, did not bind to caveolin-3, implying that caveolin binding of receptor-activated G subunits occurred only after GTP hydrolysis, which yielded a GDP-bound G subunit with high affinity for caveolin-3 (13, 15). The transient binding to caveolin was followed by reassociation of the G and G subunits and their eventual reintegration into the extracaveolar membrane. The entire cycle was completed in 40-60 min (Fig. 5). During this interval, the stoichiometry of the heterotrimeric G protein pool accessible to receptors was altered imposing a G protein-specific barrier to activation of effector enzymes (e.g. PLC-) by receptors that couple to the same G protein.

G Protein-specific Desensitization of PLC- Response-- The time course of decrease in PLC- activity in response to CCK-8 and CPA closely paralleled the time course of G binding to caveolin induced by pretreatment with ACh. At various intervals and for various concentrations of ACh, the extent of decrease in PLC- activity was correlated with the increase in G binding to caveolin. A similar G protein-specific decrease in PLC- activity was observed after sequential treatment of the cells with various agonists. Thus, activation of one receptor coupled to G_q/11 (e.g. CCK-A, NK-1, or m3 receptor) inhibited subsequent PLC- responses mediated by these receptors but not those mediated by A1 and m2 receptors. Conversely, activation of one receptor coupled to G_i3 (e.g. m2 or A1 receptor) inhibited subsequent PLC- responses mediated by these receptors but not those mediated by CCK-A, NK-1, or m3 receptors. Thus, activation of one receptor type decreased the response to another receptor type coupled to the same G protein. It is worth noting that activation of one receptor did not induce desensitization of other receptors; pretreatment of muscle cells with CCK-8, SP, or CPA, for example, had no effect on [³H]scopolamine binding, whereas pretreatment with ACh caused a large decrease in [³H]scopolamine binding, indicative of homologous desensitization of m3 and m2 receptors. PLC- activity in response to sequential stimulation with ACh decreased by about 90%, reflecting both receptor-specific (homologous) and G protein-specific (heterologous) desensitization.

Studies with caveolin-binding fragments of G_q/11 and G_i3 provided decisive evidence for selective binding of activated G to caveolin-3 and established a linkage between caveolin binding and G protein-specific heterologous desensitization of response. A caveolin-binding G_q/11 fragment that selectively blocked the binding of activated G_q/11 to caveolin-3 elicited by ACh prevented desensitization of responses mediated by other G_q/11-coupled receptors but not by G_i3-coupled receptors. Conversely, a caveolin-binding G_i3 fragment that selectively blocked the binding of activated G_i3 to caveolin-3 elicited by ACh prevented desensitization of responses mediated by other G_i3-coupled receptors but not by G_q/11-coupled receptors.

Desensitization of response by G protein binding to caveolin was not confined to G_q/11 and G_i3. Both G_i1 and G_i2 bound to caveolin-3 following activation of G_i1 by somatostatin-3 receptors, activation of G_i2 by opioid -receptors, and activation of both G_i1 and G_i2 by natriuretic peptide clearance receptors (42-44). The resultant desensitization was G protein-specific with one component reflecting G_i1 and/or G_i2 binding to caveolin-3 and the other component reflecting phosphorylation of G_i1 and/or G_i2 by PKC; only the latter was blocked by pretreatment of muscle cells with a PKC inhibitor (51).² However, neither G_q/11 nor G_i3 in smooth muscle is susceptible to phosphorylation by PKC (51); as shown in Fig. 14, desensitization of PLC- response was not affected by inhibition of PKC activity, implying that desensitization was not mediated by PKC-dependent phosphorylation of G proteins or other protein targets (receptors or effector enzymes) in the phosphoinositide signaling pathway mediated by either G_q/11 or G_i3 in smooth muscle.

In summary, this study demonstrates a role for caveolin in signal transduction that depends on its ability to bind transiently receptor-activated G protein subunits and impede reassociation of the heterotrimeric species, thereby decreasing the ability of receptors that specifically couple to these G proteins to signal effectively. The process may contribute to both homologous and heterologous desensitization of response.

	FOOTNOTES

^* This work was supported by Grant DK15564 from the NIDDK, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: P.O. Box 980711, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0711. Tel.: 804-828-8504; Fax: 804-828-2500; skarnam@hsc.vcu.edu.

Published, JBC Papers in Press, June 21, 2000, DOI 10.1074/jbc.M002194200

² K. S. Murthy and G. M. Makhlouf, unpublished results.

	ABBREVIATIONS

The abbreviations used are: GTPS, guanosine 5'-0-(-thio)triphosphate; PLC-, phospholipase C-; CPA, cyclopentyl adenosine; CCK-8, cholecystokinin octapeptide; PIP₂, phosphatidylinositol 4,5-bisphosphate; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiotide; PAGE, polyacrylamide gel electrophoresis; SP, substance P; ACh, acetylcholine; Mes, 4-morpholineethanesulfonic acid; PKC, protein kinase C; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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