Caveolin Interacts with the Angiotensin II Type 1 Receptor during Exocytic Transport but Not at the Plasma Membrane
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m212892200
ISSN1083-351X
AutoresBruce Wyse, Ian A. Prior, Hongwei Qian, Isabel C. Morrow, Susan J. Nixon, Cornelia Muncke, Teymuras V. Kurzchalia, Walter G. Thomas, Robert G. Parton, John F. Hancock,
Tópico(s)Ion channel regulation and function
ResumoThe mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R·caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R. The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R·caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R. Lipid-based sorting mechanisms play an important role in the organization of the plasma membrane into microdomains (1Brown D. London E. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 2Kurzchalia T.V. Parton R.G. Curr. Opin. Cell Biol. 1999; 11: 424-431Google Scholar, 3Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar). The biophysical properties of sphingolipids and cholesterol drive the spontaneous formation of lateral assemblies of liquid-ordered lipid rafts in a sea of liquid-disordered phospho-lipids. The biological importance of lipid rafts follows from the lateral segregation that they impose on membrane proteins. The differential distribution of plasma membrane proteins across raft and nonraft membranes in turn results in the concentration of specific groups of signaling proteins and lipids within discrete areas of the cell membrane (3Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar, 4Mineo C. Gill G.N. Anderson R.G.W. J. Biol. Chem. 1999; 274: 30636-30643Google Scholar, 5Prior I.A. Hancock J.F. J. Cell Sci. 2001; 114: 1603-1608Google Scholar, 6Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Google Scholar). This increases the efficiency and specificity of signaling events by allowing more efficient interactions between proteins and by preventing cross-talk between different pathways. Caveolae are an abundant surface feature of many mammalian cells and represent a specific subtype of lipid raft. Functionally, caveolae have been implicated in endocytosis (7Parton R.G. Joggerst B. Simons K. J. Cell Biol. 1994; 127: 1199-1215Google Scholar), potocytosis (8Anderson R.G. Kamen B.A. Rothberg K.G. Lacey S.W. Science. 1992; 255: 410-411Google Scholar), transcytosis (9Montesano R. Roth J. Robert A. Orci L. Nature. 1982; 296: 651-653Google Scholar), apical transport (10Scheiffele P. Verkade P. Fra A.M. Virta H. Simons K. Ikonen E. J. Cell Biol. 1998; 140: 795-806Google Scholar), and cholesterol balance (11Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Google Scholar). Caveolae are identified by their characteristic morphology (flask-shaped, 55–65-nm diameter pits) and the presence of integral membrane proteins, termed caveolins, of which three mammalian isoforms have been characterized (caveolin-1, -2, and -3) (12Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney G.R. Anderson R.G.W. Cell. 1992; 68: 673-682Google Scholar, 13Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Google Scholar, 14Tang Z-L. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Google Scholar). Caveolins show the characteristic biochemical features of raft-associated proteins, being associated with low density detergent-insoluble complexes that are sensitive to cholesterol depletion (15Murata M. Peranen J. Schreiner R. Wieland F. Kurzchalia T.V. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Google Scholar, 16Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Science. 1995; 269: 1435-1439Google Scholar). Caveolins are crucial structural components of caveolae. Expression of caveolin-1 in cells lacking caveolae causes de novo formation of caveolae, whereas ablation of caveolin expression causes a loss of caveolae in cultured cells and in vivo (17Fra A.M. Williamson E. Simons K. Parton R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8655-8659Google Scholar, 18Zhao Y.Y. Liu Y. Stan R.V. Fan L. Gu Y. Dalton N. Chu P.H. Peterson K. Ross Jr., J. Chien K.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11375-11380Google Scholar). The ability of caveolins to bind cholesterol and to form high molecular weight oligomeric complexes is presumably important in caveolae formation. Caveolin-1 and caveolin-2 form hetero-oligomeric complexes and are most prevalent in endothelial cells, smooth muscle cells, skeletal myoblasts, fibroblasts, and adipocytes (19Scherer P.E. Lewis R.Y. Volonte D. Engelman J.A. Galbiati F. Couet J. Kohtz D.S. van Donselaar E. Peters P. Lisanti M.P. J. Biol. Chem. 1997; 272: 29337-29345Google Scholar), whereas caveolin-3 (Cav3) 1The abbreviations used are: Cav3, caveolin-3; CSD, caveolin scaffolding domain; EGF, epidermal growth factor; EGF-R, EGF receptor; AngII, angiotensin II; AT1-R, AngII type 1 receptor; GPCR, G-protein-coupled receptor; ER, endoplasmic reticulum; HA, hemagglutinin; GFP, green fluorescent protein; BHK, baby hamster kidney; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; FRT, Fischer rat thyroid; LDL, low density lipoprotein. is exclusively present in muscle cells including cardiac myocytes and cells of the arterial vasculature (20Segal S.S. Brett S.E. Sessa W.C. Am. J. Physiol. 1999; 277: H1167-H1177Google Scholar). As well as this structural role, caveolins have also been directly implicated in interactions with signaling proteins. A juxtamembrane region of caveolin, the caveolin scaffolding domain (CSD) binds in vitro to a consensus sequence of ΦXΦXXXXΦXXΦ (where Φ is an aromatic amino acid and X is any amino acid) that is found in a large number of signaling proteins including G-proteins, conserved kinase domain IX, and elsewhere in many nonkinases (21Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Google Scholar, 22Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Google Scholar, 23Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Google Scholar). Caveolin has therefore been postulated to act as a protein scaffold for signaling proteins and to sequester them in caveolae (23Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Google Scholar), although such interactions cannot be important in tethering proteins to noncaveolar lipid rafts. The association of lipid-modified peripheral membrane proteins with lipid rafts is determined in part by the biophysical properties of the hydrophobic modification of the membrane anchor. Partitioning into rafts is favored if the lipid anchor is saturated, although in the case of prenylated and palmitoylated H-Ras activation state and additional protein sequences adjacent to the membrane anchor also influence raft association (24Prior I.A. Harding A. Yan J. Sluimer J. Parton R.G. Hancock J.F. Nat. Cell Biol. 2001; 3: 368-375Google Scholar). Less clear is how transmembrane proteins are targeted to lipid rafts. A well studied example is the epidermal growth factor receptor (EGF-R) that is extensively localized to lipid rafts in quiescent cells (25Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Google Scholar). The receptor is not lipid-modified, but recent work has shown that a 60-amino acid region in the extracellular domain of the EGF receptor, contiguous with the transmembrane domain of the receptor, is sufficient for lipid raft targeting (26Yamabhai M. Anderson R.G. J. Biol. Chem. 2002; 277: 24843-24846Google Scholar). The angiotensin II (AngII) type 1 receptor (AT1-R) is a nonpalmitoylated G-protein-coupled receptor (GPCR) that in smooth muscle cells co-immunoprecipitates with caveolin and when activated co-fractionates with caveolin on sucrose gradients (27Ishizaka N. Griendling K.K. Lassegue B. Alexander R.W. Hypertension. 1998; 32: 459-466Google Scholar). The interaction between the AT1-R and caveolin may be mediated by a CSD-binding sequence at the C terminus of the receptor (28Leclerc P.C. Auger-Messier M. Lanctot P.M. Escher E. Leduc R. Guillemette G. Endocrinology. 2002; 143: 4702-4710Google Scholar). Similar to caveolin, the AT1-R is found in many cell types including smooth and cardiac muscle cells as well as endothelial and epithelial cells (29Dinh D.T. Frauman A.G. Johnston C.I. Fabiani M.E. Clin. Sci. (Lond.). 2001; 100: 481-492Google Scholar). These observations have led to the hypothesis that an AT1-R·caveolin complex in caveolae may coordinate AngII-induced signaling (27Ishizaka N. Griendling K.K. Lassegue B. Alexander R.W. Hypertension. 1998; 32: 459-466Google Scholar). However, in contrast to the EGF-R that exits lipid rafts following ligand binding and activation (4Mineo C. Gill G.N. Anderson R.G.W. J. Biol. Chem. 1999; 274: 30636-30643Google Scholar), activated AT1-R moves into lipid rafts and/or caveolae where it transactivates the EGF-R (31Ushio-Fukai M. Hilenski L. Santanam N. Becker P.L. Ma Y. Griendling K.K. Alexander R.W. J. Biol. Chem. 2001; 276: 48269-48275Google Scholar). Recent electron microscopic studies have shown that the EGF-R is not concentrated in caveolae (32Ringerike T. Blystad F.D. Levy F.O. Madshus I.H. Stang E. J. Cell Sci. 2002; 115: 1331-1340Google Scholar), although similar data on the AT1-R are lacking. In this study we used quantitative electron microscopy to examine the surface distribution of ectopically expressed AT1-R. In addition, we show a physical interaction between the AT1-R and caveolin and use dominant-interfering mutants of caveolin to investigate interactions between caveolin and the AT1-R both at the plasma membrane and during trafficking through the exocytic pathway. We show that inactive AT1-R is found in cyclodextrin-resistant and -sensitive clusters but is not enriched in surface caveolae; treatment with AngII results in the partial relocalization of activated AT1-R into noncaveolar lipid rafts. Nevertheless, expression of mislocalized caveolin mutants, the absence of caveolin, or disrupting the formation of a caveolin·AT1-R complex has a profound effect on the trafficking of AT1-R from the ER to the plasma membrane. In addition, mouse studies indicate that caveolin is required for normal renal AT1-R expression. Together these data suggest an important chaperone role for caveolin in trafficking a transmembrane receptor to the cell surface. Plasmids—N-terminally HA-tagged AT1-R (HA-AT1-R) and C-terminally GFP-tagged AT1-R (GFP-HA-AT1-R) have been described (33Thomas W.G. Motel T.J. Kule C.E. Karoor V. Baker K.M. Mol. Endocrinol. 1998; 12: 1513-1524Google Scholar, 34Holloway A.C. Qian H. Pipolo L. Ziogas J. Miura S. Karnik S. Southwell B.R. Lew M.J. Thomas W.G. Mol. Pharmacol. 2002; 61: 768-777Google Scholar). A truncated version of the HA-AT1-R was generated by deleting 34 amino acids C-terminal to Lys325 (AT1-R,TK325) (35Thomas W.G. Baker K.M. Motel T.J. Thekkumkara T.J. J. Biol. Chem. 1995; 270: 22153-22159Google Scholar). AT1-R mutants with substitutions of key hydrophobic (Tyr302, Phe304, Phe309, and Tyr312; YFFY/A) and positively charged residues (Lys307, Lys308, Lys310, and Lys311; KKKK/Q) within the proximal C terminus (helix VIII) were generated using PCR-based site-directed mutagenesis (ExSite). The template for YFFY/A was a HA-tagged version of a previously reported AT1-R mutant (Y302A) (35Thomas W.G. Baker K.M. Motel T.J. Thekkumkara T.J. J. Biol. Chem. 1995; 270: 22153-22159Google Scholar), whereas the template for KKKK/Q was HA-AT1-R. The mutant receptors were sequenced to confirm the entire coding region and the relevant nucleotide mutations. HA- and GFP-tagged full-length Cav3 (151 residues), CavDGV (residues 54–151), Cav3-P104L (Pro → Leu substitution at residue 104), HA-tagged CavLLS (residues 75–151), and CavDGV-G55S (residues 54–154 with a Gly → Ser substitution at residue 55) are as described (36Carozzi A.J. Roy S. Morrow I.C. Pol A. Wyse B. Clyde-Smith J. Prior I.A. Nixon S.J. Hancock J.F. Parton R.G. J. Biol. Chem. 2002; 277: 17944-17949Google Scholar). For GFP-tH, GFP is targeted to the plasma membrane by the minimal H-Ras anchor, which has been described previously (37Apolloni A. Prior I.A. Lindsay M. Parton R.G. Hancock J.F. Mol. Cell. Biol. 2000; 20: 2475-2487Google Scholar). GFP-Icmt was a kind gift of Dr. Mark Philips (New York University). Caveolin-1-deficient mice have been described previously (38Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Science. 2001; 303: 2449-2452Google Scholar). Cell Culture and Transfection—Baby hamster kidney (BHK) and Human embryonic kidney (HEK) cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum. The cells were plated at 60% confluency and transfected using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. After 5 h of incubation with transfection mixture, the cells were washed with serum-free medium, and medium containing 10% calf serum was added for overnight incubation. Where indicated cholesterol depletion was performed using 1% cyclodextrin (Sigma) in DMEM for 30 min following 20 h of serum starvation as described (39Furuchi T. Anderson R.G. J. Biol. Chem. 1998; 273: 21099-21104Google Scholar). Cholesterol replenishment was carried out for 1 h using a mix of 16 μg/ml cholesterol in 0.4% cyclodextrin in DMEM as described (39Furuchi T. Anderson R.G. J. Biol. Chem. 1998; 273: 21099-21104Google Scholar). Fischer rat thyroid (FRT) cells were cultured in DMEM supplemented with 10% serum supreme at 37 °C. The cells were electroporated with expression plasmids. After 24 h, the cells were processed for confocal microscopy or biochemical analysis. Cell Fractionation—Transfected BHK cells were washed with ice-cold phosphate-buffered saline, scraped on ice into 0.3 ml of Buffer A (10 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 1 μm NaVO4, 25 mm NaF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and homogenized through a 23-gauge needle. Post-nuclear supernatants obtained by low speed centrifugation were spun at 100,000 × g at 4 °C for 30 min, and the soluble fraction (S100) and the sedimented fraction (P100) were collected. The P100 fraction, which contains cellular membranes, was rinsed and resuspended in Buffer A. Western Blotting—Protein content was measured by the Bradford reaction. 20-μg samples of the S100 and P100 fractions were separated on 10, 12, or 15% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Western blotting protein was performed using anti-HA (Babco) or anti-GFP (Roche Applied Science) antibodies. Western blots were developed using horseradish peroxidase-conjugated secondary antibodies and ECL (SuperSignal; Pierce) and quantified by phosphorimaging (Bio-Rad) as described previously (40Roy S. Lane A. Yan J. McPherson R. Hancock J. J. Biol. Chem. 1997; 272: 20139-20145Google Scholar). Confocal Microscopy—Transfected BHK and FRT cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.2% Triton X-100 for 10 min. After blocking in phosphate-buffered saline containing 3% bovine serum albumin, BHK cells were incubated with anti-HA antibody in a humidified chamber for 1 h. FRT cells were incubated with an anti-Cav3 antibody (Transduction Laboratories). The cells were then washed in phosphate-buffered saline and Cy3-coupled anti-mouse secondary antisera used to visualize protein expression. The coverslips were mounted in Mowiol for confocal microscopy (37Apolloni A. Prior I.A. Lindsay M. Parton R.G. Hancock J.F. Mol. Cell. Biol. 2000; 20: 2475-2487Google Scholar). Electron Microscopy and Statistics—Flat sheets of plasma membrane prepared from transfected BHK cells were immunogold-labeled with anti-GFP 5-nm gold and processed for image and statistical analysis exactly as described (41Prior I.A. Muncke C. Parton R.G. Hancock J.F. J. Cell Biol. 2003; 160: 165-170Google Scholar, 42Prior I.A. Parton R.G. Hancock J.F. Sci. STKE. 2003; (2003): PL9Google Scholar). Briefly, background was removed from digitized negatives using Adobe Photoshop 5.0, and the gold particle co-ordinates were determined using NIH Image 1.82. Subsequent Ripley's K-function analysis was performed using visual basic programs written into Excel macros (41Prior I.A. Muncke C. Parton R.G. Hancock J.F. J. Cell Biol. 2003; 160: 165-170Google Scholar, 42Prior I.A. Parton R.G. Hancock J.F. Sci. STKE. 2003; (2003): PL9Google Scholar). Positive deflections of the L(r)-r curve outside the 99% confidence interval for complete spatial randomness (standardized to 1 on the figures) indicate significant clustering of the gold pattern at the radius r (measured in nm) at which the deviation occurs (a more detailed explanation of the statistical theory and interpretation is given in Ref. 41Prior I.A. Muncke C. Parton R.G. Hancock J.F. J. Cell Biol. 2003; 160: 165-170Google Scholar). The gold patterns were further evaluated using a mathematical model of plasma membrane microdomains essentially as described previously (41Prior I.A. Muncke C. Parton R.G. Hancock J.F. J. Cell Biol. 2003; 160: 165-170Google Scholar). Gold particles were allocated to two types of microdomain: lipid rafts with a mean radius of 20 nm or nonrafts with a mean radius of 30 nm; in addition a fraction of particles were allocated randomly over the study area. The two types of microdomain were randomly generated over the model study area and had no fixed relationship to each other. All possible relative allocations of gold particles to raft, nonraft, or the random fraction were evaluated at the gold density achieved experimentally. Twenty Monte Carlo simulations were run for each assignment of particles. The mean K-function was calculated for each model pattern, and goodness-of-fit was evaluated by calculation of the root mean square deviation from the observed data. The model giving the lowest root mean square deviation was accepted as the best fit. Radioligand Binding—BHK cells grown in 6-well plates and HEK cells grown in 12-well plates were transfected 24 and 48 h prior to radioreceptor assay, respectively. BHK cells were incubated in a binding buffer (50 mm Tris-HCl, 100 mm NaCl, 5 mm KCl, 5 mm MgCl2, 0.6% bovine serum albumin, and 0.5 mg/ml bacitracin, pH 7.4) containing [125I]Sar1-Ile8-AngII and appropriate levels of unlabeled AngII (0–200 ng/ml) to determine specific and nonspecific binding. After 90 min of incubation, free ligand was removed by washing three times in ice-cold binding buffer. The cells were then solubilized with 0.3 m NaOH and counted on a γ counter. HEK cell surface receptor expression was examined by the receptor binding assay described above, except the radiolabel used was [3H]AngII (40 nm). Kidneys from three caveolin-1 null mice and three age-matched wild type black-6 control mice were excised, and radioreceptor assays were performed as previously described (43Wyse B. Waters M. Sernia C. Am. J. Physiol. 1993; 265: E332-E339Google Scholar). AT1-R affinity constant and expression levels were determined using Graphpad Prism (Graphpad Software Inc.). Total protein was determined by the Bradford reaction described and was used to standardize AT1-R expression levels. Immunoprecipitation—Transfected BHK cells were washed in ice-cold phosphate-buffered saline and scraped into 0.3 ml of Buffer B (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 4 mg/ml n-dodecyl β-maltoside, 0.5 mg/ml cholesteryl hemisuccinate, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 1 μg/ml aprotonin, and 1 μg/ml pepstatin). After 1 h of incubation, the cells were harvested by centrifugation (14,000 × g for 15 min). The protein content was determined by the Bradford reaction, and 1 mg of lysate was used for immunoprecipitation. After preclearing for 2 h with agarose beads, the lysates were incubated overnight with 20 μl of protein G-agarose and mouse anti-HA antisera (1:150) or 20 μl of protein A-agarose and rabbit anti-GFP antisera (1:150). The immunoprecipitates were washed twice with ice-cold washing buffer 1 (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 1 μg/ml aprotonin, and 1 μg/ml pepstatin), twice with washing buffer 2 (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, 0.1% Triton X-100, 0.05% sodium deoxycholate), and once with washing buffer 3 (50 mm Tris-HCl, pH 7.5, 0.1% Triton X-100, 0.05% sodium deoxycholate). The proteins were eluted in 55 μl of SDS-PAGE sample buffer. Activated AT1-R Localizes to Lipid Rafts but Not Caveolae— Sucrose gradient fractionation experiments have shown that stimulation with AngII causes AT1-R to shift from dense to light membranes that co-fractionate with caveolin (27Ishizaka N. Griendling K.K. Lassegue B. Alexander R.W. Hypertension. 1998; 32: 459-466Google Scholar). These data indicate that activated AT1-R associates with lipid rafts or caveolae or both, because these microdomains have similar biophysical properties. To discriminate between these possibilities, we examined the surface distribution of AT1-R in intact plasma membrane using electron microscopy. BHK cells expressing GFP-tagged AT1-R were stimulated with AngII or left untreated, and apical plasma membrane sheets were ripped off directly onto electron microscopy grids. The sheets were fixed and stained with affinity-purified anti-GFP antisera coupled directly to 5-nm gold. In parallel experiments the cells were also treated with methyl β-cyclodextrin for 30 min to disrupt cholesterol-rich lipid rafts and caveolae. Caveolae are readily identifiable by their morphology, size, and labeling for caveolin. No significant labeling of caveolae for AT1-R was evident in AngII-treated or control cells, although gold labeling was readily apparent in other areas of the plasma membrane (Fig. 1C). Inspection of the noncaveolar AT1-R gold distribution suggested that it was clustered (Fig. 1A); we therefore analyzed the gold patterns further using spatial statistics. K-function analysis reveals that the AT1-R is not randomly distributed over the cell surface but is localized to clusters with a mean radius of 20–30 nm (Fig. 1D). Cyclodextrin treatment decreased but did not cause a complete loss of clustering (Fig. 1, B and D). Stimulation with AngII for 10 min caused a significant increase in AT1-R clustering, an effect that was blocked in the presence of cyclodextrin (Fig. 1E). These results indicate that unstimulated AT1-R is resident in lipid rafts and in cholesterol-independent, nonlipid raft microdomains but that activation is accompanied by a shift of AT1-R into cyclodextrin-sensitive lipid rafts. The extent of this movement can be estimated by mathematical modeling (Fig. 1F); the observed change in AT1-R clustering would be achieved if 40% of the AT1-R in nonraft clusters moved into lipid rafts with AngII stimulation. Formation of Caveolin 3·AT1-R Complexes Is Dependent on an Intact CSD—Previous work has shown that AT1-R co-immunoprecipitates with Cav1 from vascular smooth muscle cell lysates (27Ishizaka N. Griendling K.K. Lassegue B. Alexander R.W. Hypertension. 1998; 32: 459-466Google Scholar). Fig. 2 shows identical results for AT1-R and Cav3 ectopically expressed in BHK cells. However, because Fig. 1 shows that the AT1-R is not localized to surface caveolae in these cells, we investigated where else in the cell the AT1-R·caveolin complex may form. The AT1-R protein contains a consensus site for interaction with the CSD; the sequence, Tyr302–Tyr312, is located at the C-terminal extremity of the last transmembrane domain of the receptor and the proximal portion of the C terminus (Fig. 2A). We first examined whether an intact CSD was required to form the Cav3·AT1-R complex using a series of previously characterized Cav3 mutants (Fig. 2B). CavDGV and CavLLS are N-terminal truncations at amino acids 55 and 75, respectively, such that CavDGV but not CavLLS retains the CSD. CavDGV-G55S contains a point mutation within the retained CSD. Cav3-P104L contains a naturally occurring point mutation that has been associated with mild forms of limb girdle muscular dystrophy (44Betz R.C. Schoser B.G. Kasper D. Ricker K. Ramirez A. Stein V. Torbergsen T. Lee Y.A. Nothen M.M. Wienker T.F. Malin J.P. Propping P. Reis A. Mortier W. Jentsch T.J. Vorgerd M. Kubisch C. Nat. Genet. 2001; 28: 218-219Google Scholar). The P104L point mutation does not affect the CSD but rather prevents normal trafficking of caveolin leading to Golgi accumulation (45Galbiati F. Volonte D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Google Scholar). Cell lysates from cells co-expressing each HA-tagged Cav3 mutant and GFP-tagged AT1-R were normalized for AT1-R content, immunoprecipitated with anti-GFP antiserum, and immunoblotted with anti-HA antisera. Fig. 2C shows that wild type Cav3, CavDGV, and Cav3-P104L all co-immunoprecipitate with the AT1-R. However, deletion of the CSD totally abolishes the ability of CavLLS, and mutation of the CSD significantly reduces the ability of CavDGV-G55S to form stable complexes with the AT1-R (Fig. 2, C and D). These observations show that the AT1-R and Cav3 form a stable complex and that the CSD is critically important for Cav3/AT1-R interaction. Mislocalized Cav3 Mutants Sequester the AT1-R as It Traffics to the Plasma Membrane—The preceding results suggest that the caveolin·AT1-R complex may have a biological role other than coordinating signaling at the plasma membrane. Caveolin has recently been shown to be involved in glycosylphosphatidylinositol-linked protein trafficking to the plasma membrane (46Sotgia F. Razani B. Bonuccelli G. Schubert W. Battista M. Lee H. Capozza F. Schubert A.L. Minetti C. Buckley J.T. Lisanti M.P. Mol. Cell. Biol. 2002; 22: 3905-3926Google Scholar), although no such data are available for seven transmembrane-spanning receptors. To investigate whether Cav3 was important in AT1-R trafficking, we examined the subcellular localization of the AT1-R in cells expressing mislocalized Cav3 mutants. In BHK cells transfected with HA-tagged AT1-R, either alone or with wild type Cav3, the receptor was extensively localized to the plasma membrane with a small Golgi pool (Fig. 3A). Co-expression with CavDGV caused a striking loss of plasma membrane staining for the AT1-R, which now localized extensively to areas containing lipid bodies marked by CavDGV. Lipid bodies are derived from the endoplasmic reticulum (11Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Google Scholar). CavLLS also accumulates in lipid bodies, but AT1-R localized normally to the plasma membrane in CavLLS-expressing cells. Expression of Golgi-localized Cav3-P104L,
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