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• W2013643452 abstract "In A431 cells, depletion of cholesterol with methyl-β-cyclodextrin induced an increase in both basal and epidermal growth factor (EGF)-stimulated EGF receptor phosphorylation. This increase in phosphorylation was site-specific, with significant increases occurring at Tyr845, Tyr992, and Tyr1173, but only minor changes at Tyr1045 and Tyr1068. The elevated level of receptor phosphorylation was associated with an increase in the intrinsic kinase activity of the EGF receptor kinase, possibly as a result of the cyclodextrin-induced enhancement of the phosphorylation of Tyr845, a site in the kinase activation loop known to be phosphorylated by pp60src. Cholesterol and its enantiomer (ent-cholesterol) were used to investigate the molecular basis for the modulation of EGF receptor function by cholesterol. Natural cholesterol (nat-cholesterol) was oxidized substantially more rapidly than ent-cholesterol by cholesterol oxidase, a protein that contains a specific binding site for the sterol. By contrast, the ability of nat- and ent-cholesterol to interact with sphingomyelins and phosphatidylcholine and to induce lipid condensation in a monolayer system was the same. These data suggest that, whereas cholesterol-protein interactions may be sensitive to the absolute configuration of the sterol, sterol-lipid interactions are not. nat- and ent-cholesterol were tested for their ability to physically reconstitute lipid rafts following depletion of cholesterol. nat- and ent-cholesterol reversed to the same extent the enhanced phosphorylation of the EGF receptor that occurred following removal of cholesterol. Furthermore, the enantiomers showed similar abilities to reconstitute lipid rafts in cyclodextrin-treated cells. These data suggest that cholesterol most likely affects EGF receptor function because of its physical effects on membrane properties, not through direct enantioselective interactions with the receptor. In A431 cells, depletion of cholesterol with methyl-β-cyclodextrin induced an increase in both basal and epidermal growth factor (EGF)-stimulated EGF receptor phosphorylation. This increase in phosphorylation was site-specific, with significant increases occurring at Tyr845, Tyr992, and Tyr1173, but only minor changes at Tyr1045 and Tyr1068. The elevated level of receptor phosphorylation was associated with an increase in the intrinsic kinase activity of the EGF receptor kinase, possibly as a result of the cyclodextrin-induced enhancement of the phosphorylation of Tyr845, a site in the kinase activation loop known to be phosphorylated by pp60src. Cholesterol and its enantiomer (ent-cholesterol) were used to investigate the molecular basis for the modulation of EGF receptor function by cholesterol. Natural cholesterol (nat-cholesterol) was oxidized substantially more rapidly than ent-cholesterol by cholesterol oxidase, a protein that contains a specific binding site for the sterol. By contrast, the ability of nat- and ent-cholesterol to interact with sphingomyelins and phosphatidylcholine and to induce lipid condensation in a monolayer system was the same. These data suggest that, whereas cholesterol-protein interactions may be sensitive to the absolute configuration of the sterol, sterol-lipid interactions are not. nat- and ent-cholesterol were tested for their ability to physically reconstitute lipid rafts following depletion of cholesterol. nat- and ent-cholesterol reversed to the same extent the enhanced phosphorylation of the EGF receptor that occurred following removal of cholesterol. Furthermore, the enantiomers showed similar abilities to reconstitute lipid rafts in cyclodextrin-treated cells. These data suggest that cholesterol most likely affects EGF receptor function because of its physical effects on membrane properties, not through direct enantioselective interactions with the receptor. Cholesterol is an essential component of mammalian membranes. It alters membrane fluidity, thickness, curvature, and permeability (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2557) Google Scholar, 2McMullen T.P. McElhaney R.N. Curr. Opin. Coll. Interface Sci. 1996; 1: 83-90Crossref Scopus (202) Google Scholar, 3Burger K. Gimpl G. Fahrenholz F. Cell Mol. Life Sci. 2000; 57: 1577-1592Crossref PubMed Scopus (258) Google Scholar, 4Chen Z. Rand R.P. Biophys. J. 1997; 73: 267-276Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 5Simons K. Ikonen E. Science. 2000; 290: 1721-1726Crossref PubMed Scopus (1078) Google Scholar, 6Jones D.H. Barber K.R. VanDerLoo E.W. Grant C.W. Biochemistry. 1998; 37: 16780-16787Crossref PubMed Scopus (41) Google Scholar). In addition, cholesterol is an important constituent of lipid rafts, specialized membrane microdomains that are rich in cholesterol, sphingolipids, and saturated phospholipids (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2557) Google Scholar, 6Jones D.H. Barber K.R. VanDerLoo E.W. Grant C.W. Biochemistry. 1998; 37: 16780-16787Crossref PubMed Scopus (41) Google Scholar, 7Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-41Crossref PubMed Scopus (5187) Google Scholar). Through interactions with cholesterol, the acyl chains of phospholipids in lipid rafts pack tightly together and extend fully to create a liquid-ordered phase (7Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-41Crossref PubMed Scopus (5187) Google Scholar, 8Brown R.E. J. Cell Sci. 1998; 111: 1-9Crossref PubMed Google Scholar, 9Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2065) Google Scholar). A subset of plasma membrane proteins selectively partition into the ordered environment of lipid rafts (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2557) Google Scholar, 7Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-41Crossref PubMed Scopus (5187) Google Scholar, 10Zajchowski L.D. Robbins S.M. Eur. J. Biochem. 2002; 269: 737-752Crossref PubMed Scopus (215) Google Scholar). Because of the large number of signaling proteins that are localized to lipid rafts, it has been postulated that these domains serve as regulatory platforms for some signal transduction pathways (11Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10909-10913Crossref PubMed Scopus (570) Google Scholar, 12Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar). The epidermal growth factor (EGF) 1The abbreviations used are: EGFepidermal growth factorMAPKmitogen-activated protein kinaseDMEMDulbecco's modified Eagle's mediumnat-cholesterolnatural cholesterolent-cholesterolenantiomer of cholesterolPBSphosphate-buffered salineBSAbovine serum albuminMES4-morpholineethanesulfonic acid. receptor is one of the proteins involved in signaling that is known to be enriched in lipid rafts (13Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar, 14Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 15Waugh M.G. Lawson D. Hsuan J.J. Biochem. J. 1999; 337: 591-597Crossref PubMed Scopus (129) Google Scholar). epidermal growth factor mitogen-activated protein kinase Dulbecco's modified Eagle's medium natural cholesterol enantiomer of cholesterol phosphate-buffered saline bovine serum albumin 4-morpholineethanesulfonic acid. A variety of studies have shown that EGF receptor function is affected by the levels of cholesterol, which is present at higher concentrations in lipid rafts than in the surrounding plasma membrane (16Pike L.J. Han X. Chung K.-N. Gross R. Biochemistry. 2002; 41: 2075-2088Crossref PubMed Scopus (446) Google Scholar). Depletion of cholesterol from cells leads to an increase in both basal (17Chen X. Resh M.D. J. Biol. Chem. 2002; 277: 49631-49637Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 18Ringerike T. Glystad F.D. Levy F.O. Madshus I.H. Stang E. J. Cell Sci. 2002; 115: 1331-1340Crossref PubMed Google Scholar) and EGF-stimulated (18Ringerike T. Glystad F.D. Levy F.O. Madshus I.H. Stang E. J. Cell Sci. 2002; 115: 1331-1340Crossref PubMed Google Scholar, 19Pike L.J. Casey L. Biochemistry. 2002; 41: 10315-10322Crossref PubMed Scopus (166) Google Scholar) receptor phosphorylation. The enhanced receptor tyrosine phosphorylation appears to be due to a rise in the intrinsic kinase activity of the receptor (19Pike L.J. Casey L. Biochemistry. 2002; 41: 10315-10322Crossref PubMed Scopus (166) Google Scholar). Cholesterol depletion has also been shown to result in an increase in the number of cell-surface EGF-binding sites (19Pike L.J. Casey L. Biochemistry. 2002; 41: 10315-10322Crossref PubMed Scopus (166) Google Scholar, 20Roepstorff K. Thomsen P. Sandvig K. van Deurs B. J. Biol. Chem. 2002; 277: 18954-18960Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) The increased EGF binding seems to result from an unmasking of receptors that are present on the surface of cells, but are unable to bind EGF in the presence of elevated levels of cholesterol (19Pike L.J. Casey L. Biochemistry. 2002; 41: 10315-10322Crossref PubMed Scopus (166) Google Scholar, 20Roepstorff K. Thomsen P. Sandvig K. van Deurs B. J. Biol. Chem. 2002; 277: 18954-18960Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In addition to altering the intrinsic binding and kinase activity of the EGF receptor, cholesterol also modulates signaling events directly downstream of the EGF receptor. For example, depletion of cholesterol impairs the ability of EGF to stimulate phosphatidylinositol turnover (21Pike L.J. Miller J.M. J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). By contrast, cholesterol depletion leads to the enhancement of EGF-stimulated MAPK activity (22Furuchi T. Anderson R.G.W. J. Biol. Chem. 1998; 273: 21099-21104Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). These observations suggest that cholesterol plays a significant role in modulating EGF receptor-mediated signaling. However, the molecular basis for these effects of cholesterol on EGF receptor function is not known. Cholesterol could affect EGF receptor function indirectly by influencing the physical properties of the membrane, such as thickness, fluidity, or lateral domain formation (3Burger K. Gimpl G. Fahrenholz F. Cell Mol. Life Sci. 2000; 57: 1577-1592Crossref PubMed Scopus (258) Google Scholar, 23Gimpl G. Burger K. Fahrenholz F. Biochemistry. 1997; 36: 10959-10974Crossref PubMed Scopus (404) Google Scholar). Indeed, depletion of cholesterol leads to the loss of the EGF receptor from low density lipid raft domains (19Pike L.J. Casey L. Biochemistry. 2002; 41: 10315-10322Crossref PubMed Scopus (166) Google Scholar, 21Pike L.J. Miller J.M. J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar), suggesting that some of the effects of cholesterol on EGF receptor function could be mediated through the ability of the sterol to promote lateral membrane domain formation. Alternatively, cholesterol could bind specifically to the receptor, regulating its activity through allosteric mechanisms. In this study, we further characterize the effects of cholesterol depletion on EGF receptor function and address the question of whether the effects of cholesterol are due to membrane level effects of the compound or result from specific molecular recognition of the sterol. We report here that the enhanced basal and hormone-stimulated phosphorylation of the EGF receptor that occurs upon cellular cholesterol depletion is due to a selective increase in the phosphorylation of a subset of the phosphorylatable tyrosine residues in the C-terminal tail of the receptor. Using natural cholesterol (nat-cholesterol) and its enantiomer (ent-cholesterol), we provide evidence that the effects of cholesterol on EGF receptor function are most likely due to non-enantio-selective effects of the sterol on membrane properties such as fluidity and the ability to form rafts. EGF was prepared by the method of Savage and Cohen (24Savage R.C. Cohen S. J. Biol. Chem. 1972; 247: 7609-7611Abstract Full Text PDF PubMed Google Scholar). The anti-phosphotyrosine monoclonal antibody PY20 was from Transduction Laboratories (Lexington, KY). Polyclonal antibodies against Tyr(P)845, Tyr(P)992, Tyr(P)1045, and Tyr(P)1068 of the EGF receptor were purchased from Cell Signaling Technology (Beverly, MA). A monoclonal antibody against Tyr(P)1173 of the EGF receptor was from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-flotillin monoclonal antibody was from BD Transduction Laboratories (San Diego, CA). Polyvinylidene fluoride membranes were from Osmonics, Inc. (Westborough, MA). The enhanced chemiluminescence kit was from Amersham Biosciences. The cholesterol CII and free cholesterol E assay kits were from Wako Bioproducts (Richmond, VA). Methyl-β-cyclodextrin was from Aldrich. Egg sphingomyelin and (2S,3R,4E)-2-stearoylaminooctadec-4-ene-3-hydroxy-1-phosphocholine (N-stearoylsphingomyelin) were from Avanti Polar Lipids (Alabaster, AL). All other chemicals were from Sigma. Cell Culture—A431 cells were maintained at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 7% newborn calf serum and 3% fetal calf serum. Cells were incubated overnight in DMEM containing 0.1% newborn calf serum prior to use. Chinese hamster ovary cells were maintained at 37 °C and 5% CO2 in Ham's F-12 medium containing 10% fetal calf serum. Cells were incubated overnight in Ham's F-12 medium containing 0.1% fetal calf serum prior to use. Synthesis of ent-Cholesterol—ent-Cholesterol was prepared from ent-desmosterol through a minor modification of methods described previously (25Westover E.J. Covey D.F. Steroids. 2003; 68: 159-166Crossref PubMed Scopus (27) Google Scholar). Silyl-protected ent-desmosterol was subjected to catalytic hydrogenation (300 p.s.i. H2, 15 min, platinum/carbon) and then treated to remove the silyl protecting group to give ent-cholesterol (m.p. 147.5-148 °C, [α]20 = +40.0 C (C, 1.0, in CHCl3)). Fig. 1 shows the structures of nat- and ent-cholesterol. Preparation of Cholesterol·Methyl-β-cyclodextrin Complexes—Cholesterol·methyl-β-cyclodextrin complexes were synthesized as described by Klein et al. (26Klein U. Gimpl G. Fahrenholz F. Biochemistry. 1995; 34: 13784-13793Crossref PubMed Scopus (496) Google Scholar). Briefly, 30 mg of nat- or ent-cholesterol was dissolved in 400 μl of isopropyl alcohol/chloroform (2:1, v/v). Methyl-β-cyclodextrin (1 g) was dissolved in 11 ml of phosphate-buffered saline (PBS) and heated to 80 °C with stirring. The solubilized sterol was added in small aliquots to the heated solution over 30 min. Cholesterol Depletion and Repletion—For cholesterol depletion, cells were incubated for 30 min at 37 °C in DMEM containing 50 mm HEPES (pH 7.2) and 0.1% bovine serum albumin (DMEM/BSA) and the indicated concentration of methyl-β-cyclodextrin. Control cells were incubated in the same medium lacking cyclodextrin. To replete cells with cholesterol following cyclodextrin treatment, cells were incubated at 37 °C for 30 min in DMEM/BSA containing the indicated concentration of sterol·cyclodextrin complex. For both cholesterol depletion and repletion, the tissue culture plates were swirled every 10 min to ensure continuous mixing of the components in the medium. Cholesterol Assay—Cells were washed twice with 1 ml of cold PBS, and lipids were extracted with 2 ml of hexane/isopropyl alcohol (3:2, v/v) for 1 h at room temperature. The organic extract was removed from the cell monolayer, and the solvent was removed in a SpeedVac. The lipid residue was solubilized in 1 ml of the cholesterol CII assay kit buffer solution. As reported previously (27Luker G.D. Pica C.M. Kumar A.S. Covey D.F. Piwnica-Worms D. Biochemistry. 2000; 39: 7651-7661Crossref PubMed Scopus (97) Google Scholar), color generation from nat-cholesterol standards reached a plateau after 5 min at 37 °C and remained stable through 60 min at this temperature, whereas ent-cholesterol standards required 60 min at 37 °C for complete color development. Samples were incubated for 1 h at 37 °C prior to measuring absorbance at 505 nm. After lipid extraction, the residual cell monolayers were solubilized with 10 mm sodium borate and 1% SDS. Aliquots of the solubilized material were then used for determination of total protein content using the bicinchoninic acid protein assay. Cholesterol Oxidase Assay—Assay of cholesterol oxidase was carried out utilizing the free cholesterol E assay kit. The free cholesterol E assay reagent contains cholesterol oxidase plus several additional components (peroxidase, 4-aminoantipyrene, and DEHSA (3-((3,5-dimelhoxyphenyl)ethylamino)-2-hydroxy-1-propanoic acid monosodium salt)) that permit the colorimetric detection of the hydrogen peroxide produced upon oxidation of cholesterol. For assay, 5 μg of either nat- or ent-cholesterol in complex with cyclodextrin was placed in a glass tube. One ml of the free cholesterol E assay kit buffer solution was then added. Samples were incubated at 37 °C for the indicated times. The absorbance of the samples at 600 nm was measured immediately. Equal concentrations of free cholesterol and cyclodextrin-complexed cholesterol gave equivalent absorbance measurements in this assay. Cell-surface EGF Binding—A431 cells were grown to confluence in 24-well dishes and incubated overnight in DMEM containing 0.1% fetal calf serum. After treatment to alter cholesterol content, cultures were washed with ice-cold PBS and incubated for 2 h at 4 °C in 1 ml of DMEM/BSA, 50 pm125I-EGF, and increasing concentrations of unlabeled EGF. At the end of the incubation, cells were washed three times with ice-cold PBS. Cell monolayers were dissolved in 1 ml of 1 m NaOH and counted for 125I. Data were analyzed using the LIGAND computer program (28Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7772) Google Scholar). Stimulation of Cells with EGF and Preparation of Cell Lysates—Cells in 35-mm dishes were treated to alter cholesterol content as outlined above. At the end of the incubation, EGF was added to the medium for 5 min. Cells were then washed with cold PBS and lysed by scraping the monolayers into 300 μl of radioimmune precipitation assay buffer (10 mm Tris (pH 7.2), 150 mm NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, and 5 mm EDTA) containing 1 μg/ml leupeptin, 100 μm sodium o-vanadate, 10 mmp-nitrophenyl phosphate, and 1 mm phenylmethylsulfonyl fluoride. Lysates were incubated on ice for 10 min with periodic vortexing and then clarified by centrifugation at 12000 × g for 10 min. Aliquots were then taken for determination of total protein concentration. Receptor tyrosine phosphorylation was determined by Western blotting. EGF Receptor Dephosphorylation—To assess the rate of EGF receptor dephosphorylation, cholesterol-depleted or cholesterol-repleted cells were stimulated with 1.25 nm EGF for 5 min at 37 °C. The medium was removed, and cells were washed with cold PBS. Residual cell surface-bound EGF was removed by incubating the cells twice for 2 min in 50 mm glycine and 100 mm NaCl (pH 4.0) on ice. After an additional wash with cold PBS, warmed DMEM/BSA was added, and the cells were incubated at 37 °C for the indicated times. Cells were washed with cold PBS and lysed with radioimmune precipitation assay buffer as usual. Receptor tyrosine phosphorylation was determined by Western blotting. Membrane Preparation and in Vitro Phosphorylation Assays—Cells were treated to alter cholesterol levels and then lysed by homogenization in 25 mm HEPES (pH 7.2). Membranes were pelleted by centrifugation for 10 min at 12000 × g and resuspended in 70 mm β-glycerophosphate, 250 mm NaCl, and 25% glycerol (pH 7.2). Assays were carried out in a final volume of 50 μl containing 20 mm β-glycerophosphate, 100 μm ATP, 12 mm MgCl2, 2 mm MnCl2, 20 mmp-nitrophenyl phosphate, 100 μm sodium o-vanadate, and 1 μg of membrane protein. When included, EGF was added at a final concentration of 25 nm. Membranes were incubated with growth factor for 5 min at room temperature. Assays were begun by the addition of ATP and metal ions. After incubation at 30 °C for 15 s, reactions were stopped by the addition of 50 μl of SDS sample buffer. Samples were boiled, run on a 10% SDS-polyacrylamide gel, and analyzed by Western blotting. Preparation of Lipid Rafts—All manipulations were performed at 4 °C. After appropriate treatments, one 150-mm plate of A431 cells was washed five times with PBS and scraped into 0.4 ml of 0.25 m sucrose, 1 mm EDTA, and 20 mm Tris (pH 7.8). Cells were lysed by passage through a 23-gauge needle 10 times. The lysates were sonicated five times for 15 s using a Branson 250 sonicator set at maximal power output for a microtip. After centrifugation at 1000 × g for 10 min, the post-nuclear supernatant was mixed with an equal volume of 85% sucrose in MES-buffered saline (25 mm MES (pH 6.5), 150 mm NaCl, and 2 mm EDTA) and placed in the bottom of a centrifuge tube. A 15-35% discontinuous sucrose gradient was formed above the lysate by adding sucrose-containing buffers as follows: 2 ml of 35% sucrose, 2 ml of 28% sucrose, 2 ml of 22% sucrose, and 4 ml of 15% sucrose, all in MES-buffered saline. The gradient was centrifuged for 18 h at 210,000 × g in an SW 41 rotor (Beckman Instruments). After discarding the uppermost 4 ml, the gradient was fractionated into eight 1-ml fractions. Then, 100-μl aliquots of each fraction were subjected to SDS-PAGE and analyzed by Western blotting. Western Blotting—Samples containing 25-100 μg of protein mixed with SDS sample buffer were subjected to SDS-PAGE and transferred electrophoretically to polyvinylidene fluoride membranes. The membranes were blocked with 10% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 and incubated with primary antibody for 1 h at room temperature (PY20, Tyr(P)1173, DB-1, flotillin) or overnight at 4 °C (Tyr(P)845, Tyr(P)992, Tyr(P)1045, and Tyr(P)1068). Antibody detection was carried out using enhanced chemiluminescence. Langmuir Film Balance—Water for the subphase buffer was purified by reverse osmosis, activated charcoal adsorption, and mixed-bed deionization; passed through a Milli-Q UV Plus system (Millipore Corp., Bedford, MA); and filtered through a 0.22-μm Millipak 40 membrane. Subphase buffer (pH 6.6) consisting of 10 mm potassium phosphate, 100 mm NaCl, and 0.2% sodium azide was stored under argon until used. Glassware was acid-cleaned and rinsed thoroughly with deionized water and then with hexane/ethanol (95:5). Solvent purity was verified by dipole potential measurements prior to use (29Smaby J.M. Brockman H.L. Chem. Phys. Lipids. 1991; 58: 249-252Crossref PubMed Scopus (22) Google Scholar). Final stock concentrations of sterols were determined gravimetrically using a Cahn microbalance (Model 4700), and those of sphingomyelins by lipid phosphate analysis (30Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). Surface pressure-molecular area isotherms were measured using a computer-controlled, Langmuir-type balance, described in detail previously (31Li X.-M. Momsen M.M. Smaby J.M. Brockman H.L. Brown R.E. Biochemistry. 2001; 40: 5954-5963Crossref PubMed Scopus (137) Google Scholar, 32Li X.-M. Momsen M.M. Brockman H.L. Brown R.E. Biophys. J. 2002; 83: 1535-1546Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and calibrated according to the equilibrium spreading pressures of known lipid standards (33Momsen W.E. Smaby J.M. Brockman H.L. J. Colloid Interface Sci. 1990; 135: 547-552Crossref Scopus (25) Google Scholar). The subphase was maintained at a fixed temperature using a thermostatted circulating water bath. The film balance was housed in an isolated laboratory supplied with clean air by a Bioclean air filtration system equipped with charcoal and HEPA filters. The trough was separately enclosed under humidified argon and cleaned by passage through a seven-stage series filtration setup consisting of an Alltech activated charcoal gas purifier, a LabClean filter, and a series of Balston disposable filters consisting of two adsorption (carbon) and three filter units (93 and 99.99% efficiency at 1 μm). Film balance features that contribute to isotherm reproducibility include automated lipid spreading via a modified high pressure liquid chromatography autoinjector, automated surface cleaning by multiple barrier sweeps between runs, and highly accurate reproducible setting of the subphase level by an automated aspirator. Lipids were mixed and spread (51.67-μl aliquots) from stock solutions dissolved in hexane/ethanol (95:5). Films were compressed at a rate of ≤4 Å2/molecule/min after an initial delay period of 4 min. Standard errors of the resulting force-area isotherms were routinely <2%. Site-specific Effect of Cholesterol on EGF Receptor Phosphorylation—A431 cells were treated with increasing concentrations of methyl-β-cyclodextrin for 30 min to remove cholesterol. Lysates were prepared and analyzed for receptor tyrosine phosphorylation by SDS-PAGE and Western blotting with an anti-phosphotyrosine antibody. As shown in Fig. 2A, cholesterol depletion increased basal tyrosine phosphorylation of the EGF receptor up to 11-fold. The dose of methyl-β-cyclodextrin yielding maximal stimulation was 7.5 mm when confluent cultures were used. However, when significantly subconfluent cells were used, the optimal concentration of methyl-β-cyclodextrin was 2-3-fold less than this. This indicates that it is the ratio of cells to reagent that is important rather than the absolute concentration of reagent added. In addition to enhancing basal EGF receptor phosphorylation, cholesterol depletion also increased EGF-stimulated receptor phosphorylation at all doses of EGF tested (Fig. 2B). At the maximal dose of EGF, receptor phosphorylation was enhanced from 1.2- to 2.0-fold in different experiments. The EC50 for EGF was ∼2 nm and was essentially unchanged following cholesterol depletion. Thus, the increase in EGF-stimulated tyrosine phosphorylation of the receptor following cholesterol depletion appears to be due to a change in the maximal level of phosphorylation rather than to a change in the EC50 for EGF. The increase in EGF receptor phosphorylation was associated with only a modest increase in the number of cell-surface EGF-binding sites (Fig. 3). Scatchard analysis indicated the presence of a single class of EGF-binding sites on control cells that exhibited a Kd of ∼10 nm. Cyclodextrin treatment resulted in an ∼10% increase in the number of EGF-binding sites present on A431 cells with no change in the binding affinity of EGF. The EGF receptor contains several tyrosine residues that become phosphorylated in response to EGF. To determine whether all sites were similarly affected by cholesterol depletion, EGF receptor phosphorylation was analyzed using a panel of antibodies that recognize specific phosphorylated tyrosine residues on the receptor. The data in Fig. 4 show that cholesterol depletion differentially affected the phosphorylation of individual tyrosine residues. Cholesterol depletion routinely enhanced the EGF-stimulated phosphorylation of tyrosines 845, 992, and 1173. By contrast, the level of hormone-stimulated phosphorylation of tyrosines 1045 and 1068 was relatively unaffected by removal of cholesterol. In all cases, cholesterol depletion did not significantly alter the EC50 for receptor phosphorylation. These data indicate that cholesterol depletion induces site-specific changes in phosphorylation of the EGF receptor. Comparison of the Enantiomers of Cholesterol—The effect of cholesterol depletion on EGF receptor phosphorylation could be due either to an effect of cholesterol on the physical properties of the membrane or to direct interaction of the sterol with the EGF receptor or another protein that regulates receptor phosphorylation. Theoretically, the use of cholesterol enantiomers should allow discrimination between these two possibilities. Because the physical properties of nat-cholesterol and its enantiomer, ent-cholesterol, are identical, their effects on general membrane properties should not be significantly different. However, because the two enantiomers have mirror image shapes, they should interact differently with molecules, such as proteins, that contain a stereospecific binding site for the sterol. To determine whether cholesterol affects EGF receptor function due to general membrane level effects or to direct binding to a protein, the enantiomer of cholesterol was synthesized (25Westover E.J. Covey D.F. Steroids. 2003; 68: 159-166Crossref PubMed Scopus (27) Google Scholar) and used as a tool to probe the effects of cholesterol on EGF receptor function. The ability of nat- and ent-cholesterol to interact with lipids and proteins was first compared in model systems to characterize the behavior of these two enantiomers in sterol-protein and sterol-lipid interactions. To assess the characteristics of nat- and ent-cholesterol when interacting specifically with a protein, the ability of cholesterol oxidase to use these enantiomers as substrates was compared. As shown in Fig. 5, nat-cholesterol was rapidly oxidized by cholesterol oxidase in a standard cholesterol assay system. The oxidation of nat-cholesterol was essentially complete within 30 s under these conditions and showed a t1/2 of <10 s. By contrast, ent-cholesterol was much more slowly oxidized by cholesterol oxidase under the same conditions. Approximately 30 min were required for complete oxidatio" @default.
• W2013643452 created "2016-06-24" @default.
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• W2013643452 date "2003-12-01" @default.
• W2013643452 modified "2023-10-12" @default.
• W2013643452 title "Cholesterol Depletion Results in Site-specific Increases in Epidermal Growth Factor Receptor Phosphorylation due to Membrane Level Effects" @default.
• W2013643452 cites W1498071142 @default.
• W2013643452 cites W1498171831 @default.
• W2013643452 cites W1513897103 @default.
• W2013643452 cites W1542003098 @default.
• W2013643452 cites W1564861448 @default.
• W2013643452 cites W1898709484 @default.
• W2013643452 cites W1914926542 @default.
• W2013643452 cites W1948870077 @default.
• W2013643452 cites W1963721597 @default.
• W2013643452 cites W1964295553 @default.
• W2013643452 cites W1965095341 @default.
• W2013643452 cites W1966658428 @default.
• W2013643452 cites W1986812906 @default.
• W2013643452 cites W1987898211 @default.
• W2013643452 cites W1990394752 @default.
• W2013643452 cites W1990926659 @default.
• W2013643452 cites W2004759810 @default.
• W2013643452 cites W2005192572 @default.
• W2013643452 cites W2006381992 @default.
• W2013643452 cites W2016851491 @default.
• W2013643452 cites W2022623834 @default.
• W2013643452 cites W2023286308 @default.
• W2013643452 cites W2029639174 @default.
• W2013643452 cites W2032190558 @default.
• W2013643452 cites W2037781176 @default.
• W2013643452 cites W2043813981 @default.
• W2013643452 cites W2049093635 @default.
• W2013643452 cites W2049848440 @default.
• W2013643452 cites W2053100392 @default.
• W2013643452 cites W2056681493 @default.
• W2013643452 cites W2058532748 @default.
• W2013643452 cites W2058620780 @default.
• W2013643452 cites W2059166957 @default.
• W2013643452 cites W2062421111 @default.
• W2013643452 cites W2062609965 @default.
• W2013643452 cites W2065261758 @default.
• W2013643452 cites W2065886456 @default.
• W2013643452 cites W2068908631 @default.
• W2013643452 cites W2072069399 @default.
• W2013643452 cites W2076718022 @default.
• W2013643452 cites W2077857558 @default.
• W2013643452 cites W2103950074 @default.
• W2013643452 cites W2113009216 @default.
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• W2013643452 cites W2141367941 @default.
• W2013643452 cites W2143600397 @default.
• W2013643452 cites W2144822024 @default.
• W2013643452 cites W2152430290 @default.
• W2013643452 cites W2162030013 @default.
• W2013643452 cites W2411095871 @default.
• W2013643452 cites W2950283415 @default.
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