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ORIGINAL ARTICLE

-Adrenergic Receptor Stimulation Promotes Gs Internalization through Lipid Rafts: A Study in Living Cells

Departments of Physiology & Biophysics (J.A.A., J.Z.Y., R.J.D., M.M.R.) and Psychiatry (M.M.R), University of Illinois at Chicago, College of Medicine, Chicago, Illinois

Received October 17, 2004; accepted February 9, 2005

Abstract

Upon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whether heterotrimeric G proteins also undergo agonist-induced endocytosis. To investigate the intracellular trafficking of Gs, we developed a functional Gs-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing Gs-GFP were treated with 10 µM isoproterenol, and trafficking was assessed with fluorescence microscopy. Upon isoproterenol stimulation, Gs-GFP was removed from the plasma membrane and internalized into vesicles. Vesicles containing Gs-GFP did not colocalize with markers for early endosomes or late endosomes/lysosomes, revealing that Gs does not traffic through common endocytic pathways. Furthermore, Gs-GFP did not colocalize with internalized ₂-adrenergic receptors, suggesting that Gs and receptors are removed from the plasma membrane by distinct endocytic pathways. Nonetheless, activated Gs-GFP did colocalize in vesicles labeled with fluorescent cholera toxin B, a lipid raft marker. Agonist significantly increased Gs protein in Triton X-100 –insoluble membrane fractions, suggesting that Gs moves into lipid rafts/caveolae after activation. Disruption of rafts/caveolae by treatment with cyclodextrin prevented agonist-induced internalization of Gs-GFP, as did overexpression of a dominant-negative dynamin. Taken together, these results suggest that receptor-activated Gs moves into lipid rafts and is internalized from these membrane microdomains. It is suggested that agonist-induced internalization of Gs plays a specific role in G protein-coupled receptor-mediated signaling and could enable Gs to traffic into the cellular interior to regulate effectors at multiple cellular sites.

Gs is localized primarily at the plasma membrane, where it allosterically activates its classic effector, adenylyl cyclase, resulting in the production of cAMP during receptor signaling events. It has become increasingly clear that Gs is also located in other cellular compartments. Gs has been detected in endocytic vesicles obtained from liver (Van Dyke, 2004), it associates with tubulin and the microtubule cytoskeleton in neuronal cells (Roychowdhury et al., 1999; Sarma et al., 2003), and Gs is enriched in the trans-Golgi network of rat pancreatic cells (Denker et al., 1996). Several studies have indicated other functional roles for Gs apart from activation of adenylyl cyclase, including regulation of apical transport in liver epithelia (Pimplikar and Simons, 1993), regulation of endosome fusions (Colombo et al., 1994), and controlling the trafficking and degradation of epidermal growth factor receptors (Zheng et al., 2004). How Gs is trafficked to these cellular locations and the mechanism governing its association with these subcellular compartments remain unclear.

Several previous studies have indicated that Gs undergoes a redistribution from the plasma membrane to cytosol in response to agonist stimulation (Ransnas et al., 1989; Wedegaertner and Bourne, 1994; Wedegaertner et al., 1996; Thiyagarajan et al., 2002; Yu and Rasenick, 2002; Hynes et al., 2004b); however, there are reports that have failed to see this redistribution (Jones et al., 1997; Huang et al., 1999). A fluorescent Gs-GFP fusion protein was developed by inserting green fluorescent protein into the internal sequence of Gs. This Gs-GFP fusion protein binds GTP in response to agonist, activates adenylyl cyclase, is appropriately expressed at the plasma membrane, and exhibits trafficking and signaling behavior identical with that of the wild-type Gs (Yu and Rasenick, 2002). During AR stimulation, activated Gs-GFP rapidly dissociates from the plasma membrane in living cells (Yu and Rasenick, 2002). The mechanism controlling the release of Gs from the membrane is not yet known, but it has been suggested that activated Gs is depalmitoylated and then released from the membrane (Wedegaertner and Bourne, 1994). The ultimate redistribution and putative signaling of internalized Gs is poorly understood.

We have hypothesized that, similar to receptor, Gs internalizes in response to agonist and associates with endocytic vesicles. Using real-time imaging of Gs-GFP during agonist stimulation, we demonstrate that Gs dissociates from the plasma membrane and becomes internalized in vesicles. Internalized Gs-GFP containing vesicles were derived from lipid raft domains but were not common to early or late endosomes. In addition, internalized Gs-GFP did not colocalize with ₂ARs in vesicles, suggesting that receptor and Gs traffic through distinct endocytic pathways. It is suggested that agonist-induced internalization of activated Gs may regulate endocytic trafficking and play a specific role in GPCR-mediated signaling, and it could enable Gs to traffic into the cellular interior to interact with effectors at multiple cellular sites.

Materials and Methods

Cell Culture and Transfections. MCF-7 human breast adenocarcinoma and C6 rat glioma cell lines, both of which express endogenous ₂ARs, were used for these experiments. MCF-7 cells were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum and 1% penicillin and streptomycin and were maintained in 5% CO₂ at 37°C. C6 cells were cultured in DMEM containing 4.5 g of glucose per liter, 10% calf serum supplemented with iron (Hyclone Laboratories, Logan, UT) and 1% penicillin and streptomycin and were maintained in 10% CO₂ at 37°C. Details explaining the construction of the Gs-GFP fusion protein have been described previously (Yu and Rasenick, 2002). The cDNA encoding the dominant-negative K44E dynamin 1 was originally obtained from Dr. Richard Vallee (Columbia University, New York, NY) (Herskovits et al., 1993), and it was subsequently cloned into pcDNA3.1zeo and kindly provided by Dr. Mark von Zastrow (University of California San Francisco, San Francisco, CA) (Chu et al., 1997). Both MCF-7 and C6 cells were seeded into T vision 35-mm dishes (Fisher Scientific Co., Pittsburgh, PA) for live cell imaging or onto coverslips in 12-well plates for immunofluorescence. Cells were grown to 80% confluence and were transfected for 5 h with 0.5 µg of purified Gs-GFP plasmid DNA per dish or well, using a ratio of 1:5 DNA/superfect transfection reagent (QIAGEN, Valencia, CA). Twenty-four hours after Gs-GFP transfection, cells were used for imaging experiments. Gs-GFP expression in both MCF-7 and C6 cells was semiquantified by Western blotting. Gs-GFP expression was approximately 3-fold higher than endogenous Gs expression. Coexpression of G was not required for proper membrane association of Gs-GFP. Thus, presumably, Gs-GFP uses the endogenous G for this purpose. For the dominant-negative dynamin 1 experiments, C6 cells were cotransfected for 5 h with 0.5 µg of Gs-GFP and 1.0 µgof K44E dynamin plasmid DNA per dish, using a ratio of 1:5 DNA/superfect transfection reagent. Sixteen hours after the cotransfections, cells were used for imaging experiments.

Live Cell Imaging and Immunofluorescence Microscopy. One hour before live cell imaging, complete media were replaced with serum-free DMEM supplemented with 20 mM HEPES. Cells were maintained at 37°C during the entire period of observation using a heated microscope stage (Biotechnics; Fisher Scientific). Fluorescent images were obtained using an inverted microscope equipped for fluorescent microscopy (Nikon Eclipse TE 300, excitation wavelength, 547 nm; emission wavelength, 579 nm; via high pressure Nikon Xenon XBO 100 W lamp; Nikon, Tokyo, Japan); a digital camera [RTE/CCD-1300 Y/HS (Roper Scientific, Trenton, NJ), MicroMAX camera controller (Princeton Instruments Inc., Scientific Instruments, Monmouth Junction, NJ), and Lambda 10-2 shutter (Sutter Instrument Company, Novato, CA)], and image-processing software (IPLab, Scanalytics, Fairfax, VA). All images shown were obtained using oil immersion with a 60x objective lens. Scale bars shown are 10 µm long. Cells were treated with 10 µM isoproterenol (Sigma-Aldrich, St. Louis, MO), and Gs-GFP trafficking was imaged in real time during receptor stimulation. For live cell imaging using transferrin Texas red ligand or fluorescent cholera toxin B-Alexa 555 (Molecular Probes, Eugene, OR), MCF-7 cells expressing Gs-GFP were preincubated with the probes for 20 min on ice (10 µg/ml transferrin, 400 ng/ml cholera toxin B). Cells were then washed and immediately warmed to 37°C in the presence of 10 µM isoproterenol during imaging. For imaging studies using the cholesterol chelating agent methyl--cylcodextrin (CD) (Sigma-Aldrich), C6 cells expressing Gs-GFP were preincubated with 10 mM cyclodextrin for 30 min. at 37°C, and cells were washed and subsequently imaged during treatment with 10 µM isoproterenol. To reverse the effects of CD, cholesterol was added back to cells that were initially incubated with CD. These cells were treated for 30 min with CD, washed with DMEM, and then treated with CD-cholesterol complexes for 90 min (10 µg/ml cholesterol/CD in a molar ratio of 1:6; Sigma-Aldrich) to deliver cholesterol back to the cells (Ostrom et al., 2004). These recovered cells were washed and subsequently imaged during treatment with 10 µM isoproterenol. For immunofluorescence microscopy, Gs-GFP–transfected MCF-7 cells were treated with 10 µM isoproterenol and were then fixed with 3.3% paraformaldehyde. Cells were permeabilized and blocked for 1 h with 0.5% saponin, 5% bovine serum albumin, and 1x phosphate-buffered saline. Cells were incubated with the following primary antibodies for 3 h: rabbit polyclonal anti-early endosome antigen 1 protein (EEA1) antibody (1µg/ml dilution; BD Biosciences, San Jose, CA), mouse monoclonal anti-lysosome-associated membrane protein (LAMP-1) (1 µg/ml dilution; University of Iowa Developmental Hybridoma Bank, Iowa City, IA), or with rabbit polyclonal anti-B₂AR antibody with overnight incubation (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Cells were incubated with the following secondary antibodies for 1 h: goat anti-mouse IgG rhodamine (1:100 dilution; Pierce, Rockford, IL), or goat anti-rabbit IgG rhodamine (1:100 dilution; Roche Diagnostics, Indianapolis, IN). Coverslips were mounted, and cells were imaged using fluorescence microscopy as described above. Images of live and fixed cells shown are representative of 40 to 50 cells imaged in four or more separate experiments.

Quantification of Gs-GFP Internalization. Quantification of the internalization of Gs-GFP was done as described previously (Yu and Rasenick, 2002). An individual blinded to the experimental conditions performed all measurements. The mean of gray value within the cytoplasm in fluorescence images was collected by selecting an area that corresponded to the maximal cytoplasmic region for each cell using Scion Image (Frederick, MD). Mean gray values of the Gs-GFP fluorescence in the cytoplasm were obtained and normalized per area measured. Variation of mean gray values in cytoplasm represents the change of Gs-GFP fluorescence in the interior of the cell.

Subcellular Fractionation. Confluent C6 cells in 25-cm² flasks were treated as described in the figure legends. After treatment, cells were harvested into 1 ml of phosphate-buffered saline containing 1x protease inhibitors (complete protease inhibitor cocktail; Roche Diagnostics) and homogenized with 10 strokes of a Potter-Elvehjem homogenizer, nuclei were removed by centrifugation at 1000g for 10 min, and total cellular membranes and purified cytosol were obtained by 200,000g centrifugation for 1 h using a TLA-45 rotor and Beckman TL-100 tabletop ultracentrifuge (Beckman Coulter, Fullerton, CA). Samples (10 µg) of membrane pellet and cytosol (soluble fractions) were analyzed for Gs content by immunoblotting as described below.

Isolation of Lipid Rafts/Caveolae. C6 cells were used to prepare Triton-insoluble, caveolin-enriched membrane fractions by the procedure described by Toki et al. (1999), with slight modification. C6 glioma cells were grown to confluence in 150-cm² flasks and incubated in serum-free DMEM for 1 h before all treatments. Cells were treated with 10 µM isoproterenol for 10, 30, and 60 min. Some cells were treated with 10 mM CD for 30 min to disrupt lipid rafts/caveolae or with CD followed by treatment with CD-cholesterol complexes for 90 min to redeliver cholesterol to the cells (as described above under Live Cell Imaging and Immunofluorescence Microscopy). Two flasks of cells for each treatment group were harvested into 1.0 ml of HEPES buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, and 0.3 mM phenylmethylsulfonyl fluoride) containing 1x protease inhibitor cocktail (Roche Diagnostics). Cells were homogenized with 10 strokes of a Potter-Elvehjem homogenizer, nuclei were removed by centrifugation at 1000g for 10 min, and total cellular membranes were obtained from the supernatant by 100,000g ultracentrifugation. The total membrane pellet was resuspended in HEPES buffer containing 1% Triton X-100 and incubated on ice for 30 min. The homogenate was adjusted to 40% sucrose by the addition of an equal volume of 80% sucrose prepared in HEPES buffer and placed at the bottom of an ultracentrifuge tube. A step gradient containing 30, 15, and 5% sucrose was formed above the homogenate and centrifuged at 200,000g in an SW55 rotor for 18 h. Two or three opaque bands containing the Triton X-100–insoluble floating rafts were confined between the 15 and 30% sucrose layers. These bands were removed from the gradients, diluted 3-fold with HEPES buffer, and pelleted in a microcentrifuge at 16,000g to obtain caveolin-enriched samples of lipid rafts/caveolae. To obtain samples of the nonbuoyant Triton X-100–soluble membranes, 500 µl was removed from the bottom of each ultracentrifuge tube in the 40% sucrose layer (nonbuoyant fraction). These samples were precipitated with 1 mM trichloroacetic acid in HEPES buffer for 30 min on ice followed by pelleting in a microcentrifuge. These samples of nonraft Triton X-100–soluble membrane protein and the Triton X-100–insoluble lipid rafts/caveolae were subsequently analyzed by immunoblotting.

Immunoblotting. At this point, 5 µg of each Triton X-100–soluble membrane fraction and lipid raft/caveolae fraction was subjected to SDS-polyacrylamide gel electrophoresis; 10 µg of each membrane pellet and cytosol sample was also separated by SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, which were analyzed by Western blotting. The polyvinylidene difluoride membrane was blocked for 1 h with a Tris-buffered saline/Tween 20 solution (10 mM Tris-HCl, 159 mM NaCl, and 0.1% Tween 20, pH 7.4) containing 5% dehydrated milk proteins. After three washes with Tris-buffered saline/Tween 20, membranes were incubated with polyclonal rabbit Gs antibody (1:10,000 dilution for 3 h; PerkinElmer Life and Analytical Sciences, Boston, MA) or polyclonal B₂AR antibody with overnight incubation (1:200 dilution, Santa Cruz Biotechnology). Detection of bound antibody on the blot was assessed with a horseradish peroxidase-conjugated, goat anti-rabbit IgG antibody (The Jackson Laboratory, Bar Harbor, ME) visualized by enhanced chemiluminescent detection (ECL; Amersham Biosciences, Piscataway, NJ) and quantified after scanning densitometry using ImageQuant software (Amersham Biosciences). Both the long (52 kDa) and short (45 kDa) forms of Gs were quantified together. Immunodetected Gs, ₂AR, and caveolin-1 bands were quantified, and the integrated optical density (IOD) of each band was determined and is expressed as a percentage of control. For some experiments, the original membranes were stripped with an acidic glycine buffer (100 mM glycine, pH 2.4) and reprobed using a monoclonal mouse anti-caveolin-1 antibody (1:1000 dilution overnight; BD Transduction Laboratories, Lexington, KY), followed by immunodetection. To adjust for protein loading errors, amounts of Gs (both long and short isoforms) in the Triton X-100–insoluble lipid rafts/caveolae were normalized for the level of caveolin-1 and are expressed as Gs in the caveolin-rich fraction.

Statistical Analysis. All quantified data were analyzed for statistical significance using a one-way analysis of variance followed by Student-Newman-Keuls multiple comparison test using the Prism 3.0 software package for statistical data analysis (GraphPad Software Inc., San Diego, CA). Differences were considered significant at p < 0.05.

Results

Real-Time Imaging of Gs-GFP during -Adrenergic Receptor Stimulation. C6 rat glioma and MCF-7 human breast adenocarcinoma epithelial cells are useful cell models for these studies, because both cell types can be easily transfected, and they express endogenous ₂ARs, which couple to Gs (Vandewalle et al., 1990; Manier et al., 1992). Both cell lines were transiently transfected with Gs-GFP. 24 h after transfection, cells were exposed to the AR agonist isoproterenol, and Gs-GFP trafficking was imaged in living cells during receptor stimulation. Before agonist treatment, Gs-GFP localized predominantly at the plasma membrane in C6 cells (Fig. 1A), but within 10 min after isoproterenol addition, many punctate vesicular structures appeared throughout the cytoplasm. During 10 min of treatment of both cell types, there was a marked decrease in Gs-GFP membrane localization (Fig. 1, A and B) and a contemporaneous appearance of Gs-GFP subjacent to the plasma membrane (see Supplemental Video S1).

View larger version (95K): [in this window] [in a new window]   Fig. 1. -Adrenergic receptor stimulation promotes Gs-GFP internalization in living C6 and MCF-7 cells. C6 and MCF-7 cell lines were transiently transfected with Gs-GFP. Twenty-four hours after transfection, living cells were stimulated with 10 µM isoproterenol, and real-time trafficking of Gs-GFP in response to agonist was assessed using digital fluorescence microscopy as described under Materials and Methods. Living cells were imaged in real time, and individual frames are shown before and after agonist treatment at the indicated times. A, representative C6 cell was treated for 10 min with isoproterenol. Arrows indicate regions in which Gs-GFP dissociates from the plasma membrane. B, representative MCF-7 cell treated for 10 min with isoproterenol. During agonist treatment, Gs-GFP present in membrane extensions was rapidly reorganized, and vesicles containing the protein seemed to form from these structures. Over the 8-min period of stimulation, many vesicles containing the protein were found within the cytoplasm, and these vesicles trafficked retrogradely into the cell interior. Inset images are shown 2 and 8 min after the addition of isoproterenol, and arrows indicate retrograde trafficking of a single Gs-GFP containing vesicle. See Fig. 1B Supplemental Video S1 for the real-time video of these images. Scale bars, 10 µm.

Video 1 of a representative MCF-7 cell shows that Gs-GFP at membrane extensions rapidly reorganizes to form vesicles containing this protein, and these vesicles traffic to the cell interior, indicating active endocytosis of Gs-GFP. It is noteworthy that agonist-induced removal of Gs-GFP from the plasma membrane occurred at some regions of the membrane, but not all. In C6 cells, cellular extensions of membrane enriched in Gs-GFP were repeatedly observed before agonist treatment, consistent with endogenous Gs localization in these cells (Donati et al., 2001). During receptor stimulation, Gs-GFP was removed from these structures, suggesting that internalization may occur in selective regions of the plasma membrane. Taken together, data show that during agonist stimulation of endogenous ₂ARs, activated Gs-GFP is removed from the plasma membrane, internalized by endocytosis, and localized in vesicles.

Dominant-Negative Dynamin Inhibits Agonist-Induced Internalization of Gs-GFP. The GTP binding protein dynamin 1 functions enzymatically to liberate vesicles from the plasma membrane during both clathrin-mediated and raft/caveolae-mediated endocytosis (Nichols, 2003). The K44E dominant-negative dynamin 1 is deficient in GTPase activity, rendering it nonfunctional for vesicle formation (Herskovits et al., 1993). To determine whether Gs internalization is dynamin-dependent, C6 cells were cotransfected with Gs-GFP and K44E dominant-negative dynamin 1 constructs, and living cells were imaged during isoproterenol stimulation. Figure 2 reveals that Gs-GFP is expressed predominantly at the plasma membrane of cotransfected cells before AR stimulation. During 25 min of isoproterenol exposure, Gs-GFP remained at the plasma membrane and did not internalize within vesicles or label puncta in the cellular interior. This suggests that agonist-induced internalization of Gs-GFP is dynamin-dependent.

View larger version (29K): [in this window] [in a new window]   Fig. 2. K44E dominant-negative dynamin overexpression prevents agonist-induced internalization of Gs-GFP. C6 cells were transiently co-transfected with Gs-GFP and dominant-negative K44E dynamin 1 constructs. Sixteen hours after transfection, living cells were stimulated with 10 µM isoproterenol and Gs-GFP trafficking was imaged in real time using digital fluorescence microscopy. Individual frames of Gs-GFP from a representative cell are shown before and after agonist exposure at the indicated times. Scale bar, 10 µm.

Analysis of Internalized Gs-GFP Trafficking in the Common Compartments of the Endocytic Pathway. To determine the identity and trafficking of the vesicles containing internalized Gs-GFP, antibodies against proteins commonly used as markers for early endosomes and late endosomes/lysosomes were used. MCF-7 cells expressing Gs-GFP were treated for 30 min with isoproterenol. Cells were fixed and processed for immunocytochemistry using antibodies against EEA1 or LAMP-1 to label early endosomes or late endosomes/lysosomes, respectively. As observed previously, isoproterenol treatment resulted in internalization of Gs-GFP. Fixed cells showed Gs-GFP in both vesicles and in the cytoplasm. Before agonist exposure, EEA-1 and LAMP-1 were localized to endocytic vesicles and showed a punctate localization within the cell interior. After isoproterenol treatments, internalized Gs-GFP did not colocalize with EEA1 or LAMP-1 proteins in merged images (Fig. 3, A and B). In addition, a time course of agonist treatment was performed between 5 min and 1 h, and none of the time points within this time course showed a measurable colocalization between Gs-GFP and EEA1 or LAMP-1 (data not shown). Lack of colocalization between Gs-GFP and EEA-1 or LAMP-1 suggests that Gs-GFP does not traffic through common endocytic compartments involving early or late endosomes or lysosomes.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Internalized Gs does not traffic into common endocytic compartments or colocalize with 2 adrenergic receptors. A and B, MCF-7 cells were transiently transfected with Gs-GFP, and cells were stimulated for 30 min with 10 µM isoproterenol to promote internalization. Cells were then fixed and immunocytochemistry was performed using antibodies against EEA1 or LAMP-1. A, representative MCF-7 cell showing no colocalization between internalized Gs-GFP (green) and early endosomes labeled with EEA-1 antibodies (red). B, representative MCF-7 cell reveals no colocalization between Gs-GFP (green) with late endosomes and lysosomes labeled with LAMP-1 antibodies (red). C, Gs-GFP–expressing cells were preincubated with the recycling endosome marker transferrin Texas red ligand at 4°C. Cells were warmed to 37°C in the presence of 10 µM isoproterenol, and colocalization between Gs-GFP (green) with transferrin (red) was assessed in living cells. Two representative MCF-7 cells after 15 min of isoproterenol treatment reveal no colocalization of Gs-GFP with transferrin. D, MCF-7 cells expressing Gs-GFP were stimulated for 30 min with 10 µM isoproterenol to promote internalization. Cells were fixed and processed for immunocytochemistry using a 2AR antibody. Two representative cells are shown; merged image indicates no colocalization between Gs-GFP (green) and the 2AR (red). Scale bars for all images, 10 µm.

Upon agonist binding, ₂ARs are rapidly internalized by endocytosis into clathrin-coated pits, and they traffic into recycling endosomes (Claing et al., 2002). Transferrin receptors also undergo agonist-induced endocytosis into recycling endosomes, and the transferrin receptor and its ligand are commonly used as a marker for these endosomes. To examine whether Gs-GFP internalized into recycling endosomes, colocalization of fluorescent transferrin with Gs-GFP was examined in living cells during receptor stimulation. Before agonist exposure at 4°C, both Gs-GFP and transferrin Texas red were localized at the plasma membrane. Fifteen minutes after exposure to isoproterenol, many vesicles contained Gs-GFP, but these vesicles did not colocalize with internalized transferrin (Fig. 3C, merge), indicating that Gs-GFP does not traffic into recycling endosomes.

Imaging of Internalized Gs-GFP and ₂AR after Receptor Stimulation. Because the ARs that couple to and activate Gs are rapidly internalized after agonist binding, a primary question is whether Gs accompanies the receptor during endocytosis. To test this, Gs-GFP–transfected MCF-7 cells were treated with isoproterenol over a time course, and cells were then fixed and incubated with antibody for ₂AR. It is noteworthy that MCF-7 cells express the ₂AR subtype, which mediates isoproterenol activation of Gs (Vandewalle et al., 1990; Draoui et al., 1991). Isoproterenol treatment resulted in internalization of both Gs-GFP and ₂AR, and numerous vesicles contained these proteins (Fig. 3D). Gs-GFP was also found in the cytoplasm in the paraformaldehyde-fixed cells, similar to the previous results (Fig. 3, A and B). Agonist evoked a slight overlap of Gs-GFP and ₂AR in internalized vesicles, but no obvious colocalization was observed (Fig. 3D, merge). Cells treated with agonist over a time course from 5 to 45 min were similarly examined for Gs-GFP and ₂AR colocalization, but no clear colocalization could be found at any of these time points (data not shown). This lack of colocalization of Gs-GFP and ₂AR suggests that receptor and Gs are internalized by distinct pathways.

Isoproterenol Promotes Gs-GFP and Cholera Toxin B Colocalization in Vesicles. Because Gs-GFP was not found in common endocytic compartments such as early endosomes, recycling endosomes, and late endosomes, it was hypothesized that non-clathrin–mediated endocytosis may be involved in Gs-GFP internalization. To test the idea that lipid raft/caveolae-mediated endocytosis was involved, the lipid raft marker cholera toxin B was used to label lipid rafts in living cells. Fluorescent cholera toxin B is a common marker used to investigate the trafficking of proteins during raft-mediated endocytosis in living cells (Nichols et al., 2001; van Deurs et al., 2003). It is noteworthy that it is the A subunit of cholera toxin that binds to and ADP-ribosylates Gs, whereas the B subunit acts as a ligand and binds to the lipid raft-localized ganglioside GM-1. Cholera toxin B is constitutively incorporated into cells through lipid raft/caveolae-mediated endocytosis (Orlandi and Fishman, 1998). Gs-GFP–transfected MCF-7 cells prelabeled with fluorescent cholera toxin B were subsequently treated with agonist, and living cells were visualized using digital fluorescence microscopy. Before agonist exposure, Gs-GFP strongly colocalized with cholera toxin B at the plasma membrane, presumably in lipid raft microdomains (Fig. 4, top, merge). Fifteen minutes of agonist exposure resulted in internalization of Gs-GFP and uptake of cholera toxin B (Fig. 4, bottom). Isoproterenol treatment resulted in a strong colocalization between Gs-GFP and cholera toxin B within internalized vesicles. Numerous vesicles contained both cholera toxin B and internalized Gs-GFP (arrows). This suggests that agonist-activated Gs-GFP is internalized from lipid raft domains of the plasma membrane, possibly by caveolae- or raft-mediated endocytosis.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Internalized Gs-GFP colocalizes with the lipid raft marker cholera toxin B in living MCF-7 cells. MCF-7 cells were transiently transfected with Gs-GFP, and living cells were preincubated with the lipid raft marker fluorescent cholera toxin B at 4°C as described under Materials and Methods. Cells were then warmed to 37°C in the presence of 10 µM isoproterenol, and colocalization of Gs-GFP (green) with cholera toxin B subunit (red) was assessed using digital fluorescence microscopy. Top, representative cell expressing Gs-GFP (green) and labeled with cholera toxin B (red) at 4°C before isoproterenol stimulation. Gs-GFP and cholera toxin B strongly colocalize at the plasma membrane. Bottom, merged image of two representative cells reveals strong colocalization of Gs-GFP with cholera toxin B in internalized vesicles (arrows) after isoproterenol stimulation. Scale bars, 10 µm.

Isoproterenol Stimulation Increases Gs Present in the Cytosol. Numerous studies examining the fate of Gs upon receptor activation have indicated that Gs is released into the cytosol (Ransnas et al., 1989; Wedegaertner et al., 1996; Thiyagarajan et al., 2002; Yu and Rasenick, 2002). The previous investigation using Gs-GFP demonstrated that isoproterenol treatment results in a dissociation of the fluorescent protein from plasma membrane, and the activated construct increases localization in the cytosol similar to wild-type Gs (Yu and Rasenick, 2002). To further confirm this phenomenon, C6 cells were treated with -receptor agonist for 30 min, and purified cytosol and membrane fractions were obtained and analyzed by immunoblotting for endogenous Gs content. Gs was found in the cytosol of both control and isoproterenol-treated C6 cells (Fig. 5A). Isoproterenol treatment of C6 cells increased endogenous Gs present in the cytosol by nearly 3-fold versus control. The IOD of Gs immunoblots were quantified by scanning densitometry, and data were pooled from four experiments (Fig. 5A, n = 4; Con Pellet = 1053 ± 75; Con Cytosol = 76 ± 13; ISO Pellet = 1002 ± 68; ISO Cytosol = 193 ± 22; p < 0.05, ISO Cytosol versus Con Cytosol). Increased cytosolic localization of Gsin C6 cells in response to receptor activation further confirms reports that Gs undergoes a subcellular redistribution after activation.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Isoproterenol treatment increases endogenous Gs localization in both cytosol and lipid rafts, whereas activated 2-adrenergic receptors leave lipid rafts. A, C6 glioma cells were treated with or without 10 µM isoproterenol for 30 min, and membrane and cytosolic fractions were obtained as described under Materials and Methods. The quantity of endogenous Gs in the membrane pellet (P) and in cytosolic soluble fractions (S) was determined by Western blotting. A representative blot (n = 4) of Gs protein indicates that isoproterenol increases the amount of Gs present in the cytosol without changing the total amount of Gs. B, agonist stimulation increases Gs localization in rafts. C6 glioma cells were treated with or without isoproterenol for 30 min. Detergent-insoluble lipid rafts/caveolae (Raft) as well as nonraft detergent-soluble membranes (TXSol) were obtained by sucrose density gradient fractionation (see Materials and Methods). A representative blot of Gs protein is shown (top); the same blot was reprobed for the lipid raft/caveolae marker protein caveolin-1 (bottom). The figure is a quantification of Gs protein obtained by scanning densitometry and is presented as a percentage of control (n = 3). C, time course of isoproterenol treatment and Gs localization in the lipid raft/caveolae membrane fractions. C6 glioma cells were stimulated with isoproterenol for 10, 30, and 60 min, and rafts were obtained as in B. A representative blot of Gs protein in lipid rafts after isoproterenol treatment is shown (top), as well as caveolin-1 protein (bottom). The figure shows the percentage of change in Gs protein above control in the lipid raft/caveolae membrane fractions from four independent experiments. D, 2-adrenergic receptors leave lipid rafts after agonist stimulation. C6 cells were treated, membrane fractions were obtained as in B, and immunoblotting was performed for the 2AR. A representative blot of 2AR protein in these fractions before and after isoproterenol treatment is shown (top), along with the same blot reprobed for caveolin-1 protein (bottom). The figure is a quantification of 2AR protein obtained by scanning densitometry and is presented as a percentage of control (n = 3). Data are represented as the mean ± S.E.M. *, p < 0.05 versus control. Approximate molecular mass markers are shown in kilodaltons.

Analysis of Gs and ₂AR Localization in Lipid Rafts/Caveolae. Colocalization of Gs-GFP with the lipid raft marker cholera toxin B (Fig. 4) suggests that lipid rafts may play an important role in agonist-induced internalization of Gs. To assess biochemically the localization of endogenous Gs in these membrane domains, C6 cells were treated with or without isoproterenol and Triton X-100 detergent-resistant raft/caveolae membranes, and Triton X-100–soluble nonraft membranes were isolated by sucrose density gradient ultracentrifugation as described under Materials and Methods. The amount of endogenous Gs in these membranes was determined by immunoblotting. Immunoblots were probed for both Gs (Fig. 5B, top) and the protein caveolin-1 (Fig. 5B, bottom), which is a positive marker found exclusively in lipid rafts/caveolae. Gs was found in both detergent-resistant raft/caveloae fractions and also Triton X-100–soluble membrane fractions. Thirty minutes of isoproterenol treatment resulted in a significant increase in Gs protein present in the lipid raft/caveolae fractions and a concomitant decrease in the Triton X-100–soluble membrane (nonraft) fractions (Fig. 5B). A time course of isoproterenol treatment was also performed, and the percentage of change in Gs protein in the lipid raft/caveolae fractions was determined (Fig. 5C). Isoproterenol treatment resulted in increased Gs localization in rafts by approximately 30% above control levels within 10 min (Fig. 5C). This agonist-induced increase of Gs in rafts/caveolae indicates that Gs moves into these membrane microdomains subsequent to agonist activation.

Likewise, the raft and nonraft membrane localization of the ₂AR was determined before and after agonist stimulation. The polyclonal ₂AR antibody recognized two bands between the 47- and 76-kDa markers; the lowest band was estimated to be approximately 62 kDa. ₂AR immunoreactivity seemed enriched in the Triton X-100–soluble membranes, with a lesser portion detected in the caveolin-enriched raft/caveloae fractions (Fig. 5D). However, after 30 min of isoproterenol treatment, ₂ARs were significantly decreased in the raft/caveolae fractions by nearly 80% (Fig. 5D). This decrease in ₂AR localization in rafts/caveolae is consistent with previous reports in other cell types demonstrating that activated ₂ARs leave these microdomains (Rybin et al., 2000; Ostrom et al., 2001). Taken together, these data demonstrate that agonist stimulation results in subtle shifting of Gs and ₂AR in or out of raft/caveloae membranes during signaling.

Depletion of Membrane Cholesterol Prevents Gs-GFP Internalization. Increased localization of Gs in lipid raft/caveolae fractions after isoproterenol exposure suggests that these microdomains are important for Gs trafficking. Depletion of cholesterol from cell membranes using the chelating agent CD is commonly used to inhibit raft/caveolae-mediated endocytosis (Nichols, 2003). Exposure of C6 cells to 10 mM CD for 30 min resulted in a profound decrease in the amount of endogenous Gs located in lipid rafts/caveolae and increased the amount of Gs present in the soluble nonraft membranes (Fig. 6A, top). Depletion of cholesterol with CD decreased both long and short forms of Gs present in raft/caveolae fractions, whereas the amount of caveolin-1 was not affected (Fig. 6A, bottom). These results show that removing cholesterol from cell membranes results in a significant depletion of Gs present in lipid rafts/caveolae. To ensure that CD was not toxic, cholesterol complexes were added back to CD-treated cells as described under Materials and Methods. Adding cholesterol back to cells partially restored Gs localization to the raft/caveloae fractions (Fig. 6B).

View larger version (51K): [in this window] [in a new window]   Fig. 6. Disruption of lipid rafts prevents Gs-GFP internalization. A, C6 cells were incubated with or without 10 mM CD for 30 min or with cyclodextrin followed by CD-cholesterol complexes (CD + CHOL) for 90 min to restore cholesterol back to the cells. 1% Triton X-100 –soluble membranes (TXSol) and -insoluble rafts/caveolae (Raft) were obtained as described in Fig. 5. A representative immunoblot shows endogenous Gs protein in these fractions (top) and the lipid raft/caveolae marker caveolin-1 (bottom). Approximate molecular mass markers are shown. B, quantification of Gs protein by scanning densitometry and presented as a percentage of control (n = 3). C, top, C6 cells expressing Gs-GFP were treated with CD for 30 min, washed, and preincubated with transferrin Texas red ligand at 4°C. Cells were warmed to 37°C in the presence of 10 µM ISO, and Gs-GFP and transferrin trafficking were assessed in living cells using fluorescence microcopy. Images of a representative C6 cell reveals that CD treatment prevents agonist-induced internalization of Gs-GFP but not internalization of transferrin. C, bottom, C6 cells expressing Gs-GFP were initially treated with CD and then with CD-cholesterol complexes to restore lipid rafts. Images of a representative C6 cell reveal that CD effects can be reversed by adding cholesterol back to the cells because isoproterenol treatment of these cells restored Gs-GFP internalization. D, quantification of Gs-GFP internalization in C6 cells. The gray value intensity of Gs-GFP present in the cytoplasm of cells was measured using NIH Image software as described under Materials and Methods. Data are from images of cells pooled from 10 independent experiments (n = 10). Scale bars, 10 µm. Data are represented as the mean ± S.E.M. *, p < 0.05 versus control.

To investigate whether CD can block Gs-GFP internalization, C6 cells expressing Gs-GFP were exposed to 10 mM CD for 30 min followed by isoproterenol stimulation during live cell imaging. A representative C6 cell shown in Fig. 6C demonstrates that agonist-induced internalization of Gs-GFP is prevented by cyclodextrin treatments. Gs-GFP remained localized on the plasma membrane during agonist stimulation, and Gs-GFP was not found in vesicles or punctate structures. Although CD treatment prevented Gs-GFP internalization, it did not inhibit clathrin-mediated endocytosis of transferrin (Fig. 6C, top). In similar experiments, C6 cells expressing Gs-GFP were initially treated with CD, and then cholesterol was delivered back to cells in the form of CD-cholesterol complexes. Images of a representative C6 cell demonstrate that CD effects are reversed when cells are provided cholesterol complexes to restore lipid rafts/caveloae (Fig. 6C, bottom). Fluorescent images of C6 cells pooled from 10 independent experiments were quantified for Gs-GFP internalization after treatments. These data demonstrate that disrupting rafts with cyclodextrin prevents agonist-induced internalization of Gs-GFP, and this inhibition is reversible if cholesterol is delivered back to C6 cells. The blockade of Gs-GFP internalization by inhibition of raft/caveolae endocytosis suggests that Gs endocytosis is carried out through lipid rafts/caveolae.

Quantification of Gs-GFP Internalization in MCF-7 Cells. To quantitatively assess Gs internalization in MCF-7 cells, Gs-GFP–expressing cells were treated with isoproterenol for 30 min, fluorescent images were obtained, and the gray value intensity of Gs-GFP present in the cytoplasm of cells was measured using NIH image software as described under Materials and Methods. It is worth noting that this method of quantifying internalization detects the intracellular signal of Gs-GFP, regardless of whether the fluorescence is cytoplasmic or vesicular in nature. Isoproterenol treatment significantly increased Gs-GFP internalization versus control cells (Fig. 7). In contrast, cells cotransfected with Gs-GFP and dominant-negative K44E dynamin 1 did not show a significant internalization in response to agonist. Likewise, cells pretreated with the lipid raft inhibitor methyl--cyclodextrin also did not show agonist-mediated internalization of Gs-GFP. Quantitative data are from images of MCF-7 cells pooled from 10 independent experiments. This quantitative assessment suggests that both Gs-GFP internalization in vesicles and redistribution of Gs-GFP into the cytoplasm require intact lipid rafts/caveolae and dynamin-dependent endocytosis.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Quantification of Gs-GFP internalization. MCF-7 expressing Gs-GFP were treated with or without isoproterenol for 30 min, and fluorescent images were obtained. The gray value intensity of Gs-GFP present in the cytoplasm of cells was measured using NIH Image software as described under Materials and Methods. ISO treatment significantly increases Gs-GFP in the cytoplasm of cells, but this internalization is prevented when cells are cotransfected with dominant-negative K44E dynamin 1 (K44E Dyn) or in cells treated with the lipid raft inhibitor CD. Data are from images of MCF-7 cells pooled from 10 independent experiments (n = 10). *, p < 0.05 versus control.

Discussion

To assess the real-time trafficking of Gs during signal transduction, we used the well-established approach of visualizing a GFP fusion protein, providing a convenient method for examining G protein trafficking in real time (Janetopoulos and Devreotes, 2002; Yu and Rasenick, 2002; Hynes et al., 2004a). We have purposefully chosen to study Gs trafficking by expressing Gs-GFP at low levels in C6 and MCF-7 cells that express only endogenous ₂ARs. This model enables Gs-GFP to become activated by only endogenous ARs, and we consider this approach preferable to overexpressing receptors. Real-time imaging of Gs-GFP demonstrates that isoproterenol treatment results in a removal of Gs-GFP from the plasma membrane and internalization of the protein within vesicles (Fig. 1A and Supplemental Movie S1). Agonist-induced internalization of Gs-GFP seems to be dynamin 1-dependent, because overexpression of the K44E dominant-negative dynamin mutant prevented Gs-GFP internalization (Figs. 2 and 7). Data also demonstrate that Gs-GFP redistributes into the cytoplasm after receptor stimulation (Figs. 3 and 4), which is consistent with previous findings about both wild-type Gs and Gs-GFP (Yu and Rasenick, 2002). Note that both endogenous Gs and Gs-GFP have an identical cellular distribution, and previously, both identically redistributed in response to isoproterenol (Yu and Rasenick, 2002). In addition, agonist significantly increased the content of endogenous Gs in the cytosol of C6 cells (Fig. 5A), and this supports the many studies demonstrating a cytosolic redistribution of activated Gs.

We were surprised to find that markers for early and recycling endosomes, as well as late endosomes/lysosomes, did not colocalize with internalized vesicles containing Gs-GFP (Fig. 3, A–C). Because neither EEA-1 nor transferrin colocalized with Gs-GFP, it is unlikely that early endosomes or recycling endosomes are involved in trafficking of Gs during internalization. Consistent with these results, internalized vesicles containing Gs-GFP did not colocalize with endocytosed ₂ARs (Fig. 3D), which are known to traffic into early and recycling endosomes (Claing et al., 2002). Lack of colocalization of internalized Gs-GFP with ₂ARs agrees with previous results showing that internalized Gs does not colocalize with the receptors in endosomes (Wedegaertner et al., 1996; Hynes et al., 2004b). These results collectively suggest that internalized Gs does not traffic in common compartments of the endocytic pathway.

Both lipid rafts and caveolae are cholesterol- and glycolipid-rich microdomains of the plasma membrane involved in a mode of endocytosis distinct from classic clathrin-mediated endocytosis. Because internalized Gs-GFP did not colocalize with markers for common endocytic compartments, we investigated the potential involvement of lipid rafts. Using the fluorescent marker cholera toxin B, which binds to and is internalized from rafts, microscopy demonstrates that internalized Gs-GFP strongly colocalizes with cholera toxin B in vesicles in living cells (Fig. 4), suggesting that Gs is internalized from raft microdomains. It is worth noting cholera toxin B may also label compartments such as early endosomes in some cell types (Torgersen et al., 2001), but cholera toxin B is endocytosed predominantly by non-clathrin–mediated endocytosis (Orlandi and Fishman, 1998; Nichols et al., 2001; van Deurs et al., 2003). Colocalization of cholera toxin B with Gs-GFP in internalized vesicles strongly suggests that lipid rafts are involved in Gs internalization.

To further support this observation, biochemical studies revealed that isoproterenol treatment of C6 cells significantly increased the level of endogenous Gs located in Triton X-100 detergent-resistant raft/caveloae fractions (Fig. 5, B and C). The finding that in unstimulated cells, Gs is present in rafts/caveolae is consistent with previous studies (Toki et al., 1999; Oh and Schnitzer, 2001). Increased localization of Gs in raft/caveolae fractions suggests that Gs moves into these membrane domains during receptor stimulation, in which it may subsequently become internalized. In contrast to this, it seems that activated ₂ARs leave the lipid raft/caveloae microdomains (Fig. 5D), data that are consistent with reports shown in the cardiomyocyte (Rybin et al., 2000; Ostrom et al., 2001). There seems to be cellular heterogeneity concerning ₂AR compartmentalization. ₂ARs seem to be distributed evenly between raft and nonraft fractions in cardiomyocytes; however, in other tissues such as vascular smooth muscle or airway epithelia, ₂ARs are largely excluded from rafts (Ostrom and Insel, 2004). Our data show that in C6 glioma cells, ₂ARs are found predominantly in nonraft fractions (Fig. 5D). Although the fractions examined do not account for all Gs and ₂AR, Gs seems enriched in the raft/caveolae domains, whereas ₂ARs are weighted to nonrafts. Considering that the ratio of ₂AR to Gs is 1:100 in C6 cell membranes, which we have calculated (Manier et al., 1992; Toki et al., 1999), this would increase the ratio of receptor to Gs in the nonraft regions. It is unclear how these ratio differences of Gs to ₂AR in the membrane domains contribute to AR signaling. The increased association of Gs with rafts but removal of ₂ARs from rafts during signaling further supports the hypothesis that Gs internalizes and traffics distinctly from the ₂AR.

Additional evidence supporting the hypothesis that rafts mediate Gs internalization can be found from the studies using methyl--cyclodextrin. Incubation of C6 cells with cyclodextrin resulted in a profound decrease in Gs located in rafts/caveolae, demonstrating the importance of cholesterol in targeting Gsto these microdomains (Fig. 6, A and B). In C6 cells preincubated with cyclodextrin before isoproterenol treatment, Gs-GFP internalization was blocked without affecting transferrin internalization (Fig. 6C). It is noteworthy that the inhibitory effects of cyclodextrin were reversed by adding cholesterol back to cells, indicating that cyclodextrin treatments are not toxic to the cells. It is noteworthy that in certain conditions, cyclodextrin has also been reported to inhibit clathrin mediated endocytosis in some cell lines (Subtil et al., 1999). However, because cyclodextrin did not prevent endocytosis of transferrin, it is unlikely that clathrin-mediated endocytosis was inhibited by cyclodextrin in these experiments. Similar to C6 cells, cyclodextrin also prevented Gs-GFP internalization in MCF-7 cells treated with isoproterenol (Fig. 7). These results support the conclusion that AR stimulation promotes Gs movement into lipid rafts and that these microdomains are necessary for Gs internalization.

A summary of experimental results and a working model for Gs internalization is illustrated and described in Fig. 8. This model proposes that Gs becomes internalized within vesicles derived from lipid rafts and that Gs trafficking is distinct from the ₂AR.

View larger version (26K): [in this window] [in a new window]   Fig. 8. A model for agonist-induced internalization of Gs. Upon isoproterenol binding to the 2AR, Gs exchanges GDP for GTP, resulting in dissociation of the G proteins from the receptor, and Gs becomes activated. Within seconds, the receptor becomes phosphorylated by G protein-coupled receptor kinases, and through interactions with -arrestin, the receptor is recruited into clathrin-coated pits and trafficked into early endosomes in which ligand is removed by acidic pH (Claing et al., 2002). The receptor traffics into classic endosomes containing EEA-1 and transferrin markers. After agonist activation, the 2AR leaves lipid raft/caveolae microdomains (Fig. 5D). In contrast to this, activated Gs increases its association with lipid raft microdomains (Fig. 5, B and C), and it is internalized within distinct vesicles derived from those domains. These vesicles containing Gs positively label with the lipid raft marker cholera toxin B (Fig. 4). Gs internalization from lipid rafts is dynamin 1-dependent (Figs. 2 and 7) and can be inhibited by the lipid raft-disrupting drug cyclodextrin (Figs. 6 and 7). After receptor activation, Gs is also released into the cytosol (Fig. 5A), and this release may occur from either lipid rafts at the plasma membrane or from internalized vesicles. Taken together, this model suggests that activated Gs becomes internalized within vesicles derived from lipid rafts and that Gs trafficking is distinct from the 2AR.

The agonist-mediated internalization and trafficking of ARs is a well-described phenomenon; however, relatively little is understood about the mechanisms regulating heterotrimeric G protein internalization. We have focused on the trafficking of Gs in this study, but very recent work demonstrates that G is also internalized in response to agonist in human embryonic kidney 293 cells (Hynes et al., 2004b). Internalization of activated G proteins adds a substantial complexity to the regulated signaling of -adrenergic receptors, one that requires proper trafficking of both receptors and their cognate G proteins to maintain signaling fidelity. Recent experiments have shown that protein kinase A-phosphorylated ₁ARs will preferentially internalize through caveolae/rafts (Rapacciuolo et al., 2003). Although cells studied in this investigation express the ₂AR subtype, in future experiments, it will be instructive to investigate whether ₁ARs traffic together with internalized Gs from rafts/caveloae.

Lipid rafts/caveolae are typically thought of as membrane microdomains that spatially organize molecules to facilitate GPCR-mediated signaling. However, this assumption probably depends on which G protein and receptor pathway are involved. Recently, Roth and coworkers demonstrated in C6 cells that 5-hydroxytryptamine-2A/Gq-coupled receptor pathways are dependent on caveolae and interactions with caveolin-1, suggesting that caveolae promote 5-hydroxytryptamine-2A/Gq signaling (Bhatnagar et al., 2004). In contrast to this, results in this report suggest that Gs in lipid rafts/caveolae may be removed from membrane signaling cascades. Treatment of C6 cells with antidepressant drugs results in a removal of Gs from rafts/caveolae and an increase in cAMP synthesis, supporting the concept that shifting Gs out of raft domains enhances cAMP signaling (Toki et al., 1999; Donati et al., 2001). Two previous studies in which lipid rafts/caveolae were disrupted by cyclodextrin revealed that depletion of rafts significantly increased isoproterenol-stimulated cAMP production (Rybin et al., 2000; Miura et al., 2001). Increased cAMP production in cells depleted of rafts/caveolae is consistent with the notion that these domains are effective in silencing cAMP production. Thus, isoproterenol-induced movement of Gs into lipid rafts and its subsequent internalization may be involved in modulating cAMP production; however, this has yet to be confirmed.

Internalization could also enable activated Gs to interact with effectors at multiple intracellular sites. Several studies have indicated that Gs regulates endocytic trafficking. Gs seems to be involved in the regulation of apical transport in liver epithelia (Pimplikar and Simons, 1993), and antibodies against Gs prevent the fusion of endosomal vesicles (Colombo et al., 1994). Recently, Gs has been implicated in regulating the trafficking and degradation of epidermal growth factor receptors through interactions on endosomal vesicles (Zheng et al., 2004). Isoproterenol-induced internalization of Gs from rafts is consistent with these findings and may be an event enabling activated Gs to traffic into the cellular interior to regulate endocytic pathways. Finally, Gs has also been shown to associate with cytoskeletal elements, and Gs is capable of activating the GTPase of tubulin and increasing microtubule dynamics (Roychowdhury et al., 1999; Sarma et al., 2003). Thus, internalization of Gs and association with the microtubule cytoskeleton could be a mechanism facilitating agonist-mediated cell-shape changes. In future studies, it will be informative to investigate the roles of both the actin and microtubule cytoskeletons in vesicular trafficking of Gs.

In summary, this report reveals that activated Gs-GFP translocates away from the plasma membrane by endocytosis and that Gs trafficking is separate from ₂ARs. Activated Gs increases its localization in lipid rafts/caveolae during agonist treatment, and internalization occurs from lipid raft microdomains of the plasma membrane that is dynamin 1-dependent. It is suggested that agonist-induced internalization of Gs and association with vesicles may alter cAMP production and enable Gs to participate in intracellular signaling events.

Acknowledgements

We thank Drs. R. Vallee and M. von Zastrow for the generous gift of material. We thank Dr. Karen Colley for valuable advice and discussion. We thank the members of the Rasenick laboratory for critical reading of the manuscript.

Footnotes

This work was supported by research grant MH39595 from the National Institutes of Health (to M.M.R.), and by National Institutes of Health training grant T32-HL07692 (to J.A.A.). The study has been presented previously in preliminary form at the 43rd Annual Meeting of the American Society for Cell Biology; 2003 Dec 13–17; San Francisco, California, and at the 17th Annual Meeting of the Great Lakes Chapter of the American Society for Pharmacology and Experimental Therapeutics; 2004 Jun 3; Maywood, Illinois.

J.A.A. and J.Z.Y. contributed equally to this work.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.104.008342.

ABBREVIATIONS: GPCR, G protein-coupled receptor; GFP, green fluorescent protein; AR, -adrenergic receptor; ISO, isoproterenol; EEA1, early endosome antigen 1 protein; LAMP-1, lysosome-associated membrane protein 1; CD, methyl--cyclodextrin; DMEM, Dulbecco's modified Eagle's medium; IOD, integrated optical density.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Mark M. Rasenick, Department of Physiology and Biophysics (MC 901), College of Medicine, University of Illinois at Chicago (UIC), 835 South Wolcott Avenue, Chicago, IL 60612–7342. E-mail: raz{at}uic.edu

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