The CD20 Calcium Channel Is Localized to Microvilli and Constitutively Associated with Membrane Rafts
2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês
10.1074/jbc.m400525200
ISSN1083-351X
AutoresHaidong Li, Linda M. Ayer, Maria J. Polyak, Cathlin M. Mutch, Ryan J. Petrie, Laura Gauthier, Neda Shariat, Michael J. Hendzel, Andrew Shaw, Kamala D. Patel, Julie P. Deans,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoCD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein. CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein. CD20 is a B cell-specific tetraspan protein that assembles into oligomeric complexes and forms or regulates a store-operated calcium entry channel that is responsive to B cell receptor (BCR) 1The abbreviations used are: BCR, B cell receptor; mAb, monoclonal antibody; EM, electron microscopy; PBMC, peripheral blood mononuclear cells; GFP, green fluorescence protein; A488, Alexa 488; CTB, cholera toxin subunit B; PFA, paraformadehyde; GM1, Galβ1, 3GalNAcβ1,4(NeuAcα2,3)Galβ1,4Glcβ1,1-ceramide; MBC, methyl-β-cyclodextrin; NaN3, sodium azide; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid. 1The abbreviations used are: BCR, B cell receptor; mAb, monoclonal antibody; EM, electron microscopy; PBMC, peripheral blood mononuclear cells; GFP, green fluorescence protein; A488, Alexa 488; CTB, cholera toxin subunit B; PFA, paraformadehyde; GM1, Galβ1, 3GalNAcβ1,4(NeuAcα2,3)Galβ1,4Glcβ1,1-ceramide; MBC, methyl-β-cyclodextrin; NaN3, sodium azide; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid. signaling (1Li H. Ayer L.M. Lytton J. Deans J.P. J. Biol. Chem. 2003; 278: 42427-42434Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). CD20 is also an effective target for in vivo depletion of malignant or autoimmune B cells using monoclonal antibodies (mAbs), which can activate apoptotic signaling pathways and mediate complement-mediated cytoxicity potentially through mechanisms involving cholesterol- and sphingolipid-rich membrane microdomains known as lipid rafts (2Deans J.P. Li H. Polyak M.J. Immunology. 2002; 107: 176-182Crossref PubMed Scopus (210) Google Scholar, 3Maloney D.G. Smith B. Rose A. Semin. Oncol. 2002; 29: 2-9Crossref PubMed Scopus (308) Google Scholar, 4Cragg M.S. Morgan S.M. Chan H.T. Morgan B.P. Filatov A.V. Johnson P.W. French R.R. Glennie M.J. Blood. 2003; 101: 1045-1052Crossref PubMed Scopus (323) Google Scholar). Rafts are thought to function in part as platforms for signaling from those receptors with properties that allow their access to the tightly packed lipid raft environment, which otherwise excludes most membrane proteins (5Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2043) Google Scholar, 6Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5111) Google Scholar, 7Pierce S.K. Nat. Rev. Immunol. 2002; 2: 96-105Crossref PubMed Scopus (275) Google Scholar). The operational criteria for assigning raft association of a protein are insolubility in nonionic detergents and buoyancy on density gradients. The detergent best characterized for raft isolation is Triton X-100, and we showed previously that antibodies induce translocation of CD20 from the soluble fraction of Triton X-100 cell lysates into the buoyant insoluble fraction, consistent with its induced association with rafts (8Deans J.P. Robbins S.M. Polyak M.J. Savage J.A. J. Biol. Chem. 1998; 273: 344-348Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). However, raft association of some proteins can only be demonstrated using very low concentrations of Triton X-100 or other nonionic detergents (9Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (270) Google Scholar, 10Hostager B.S. Catlett I.M. Bishop G.A. J. Biol. Chem. 2000; 275: 15392-15398Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 11Schuck S. Honsho M. Ekroos K. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2003; Google Scholar), and we recently found that unligated CD20, although soluble in 1% Triton X-100, was insoluble in 1% Brij 58 (1Li H. Ayer L.M. Lytton J. Deans J.P. J. Biol. Chem. 2003; 278: 42427-42434Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Brij 58-insoluble CD20 localized to cholesterol-dependent, buoyant fractions on sucrose density gradients. Importantly, deletion of a short membrane-proximal cytoplasmic sequence, previously shown to be essential for efficient translocation into Triton-resistant rafts (12Polyak M.J. Tailor S.H. Deans J.P. J. Immunol. 1998; 161: 3242-3248PubMed Google Scholar), also prevented the constitutive association of CD20 with Brij 58 buoyant fractions. CD20 thus appears to be an example of a raft-associated protein that is Triton-soluble. What, then, is the meaning of antibody-induced translocation into Triton-resistant rafts? Here, we first extend our observations on the constitutive nature of CD20-raft association and then explore the effects of antibody ligation on cell surface distribution and mechanisms of antibody-induced Triton-insolubility of CD20.Our results show that antibody binding does not induce a detectable alteration in cell surface distribution of CD20, which in the absence of antibody binding is already distributed unevenly on the B cell surface. Transmission electron microscopy (EM) demonstrated that CD20 was localized to membrane protrusions, or microvilli. Anti-CD20 mAbs differ dramatically in their ability to induce Triton insolubility of CD20, and we show here that these differences cannot be attributed to the antibody isotype or to the amount of antibody bound per cell but are epitope-dependent. Translocation into Triton-resistant rafts is shown to be independent of the availability of ATP, kinase activity, actin polymerization, and the cross-linking effects of antibodies. Together, these data are consistent with the interpretation that CD20 is constitutively associated with lipid rafts on microvilli and that antibody engagement increases the affinity of the association through an intrinsic mechanism, such as a conformational change in the CD20 protein.EXPERIMENTAL PROCEDURESCells—Ramos and BJAB B cells were maintained in culture in RPMI 1640 plus 7.5% fetal bovine serum. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using lymphocyte separation medium (ICN Biomedicals, Aurora, OH). Stable transfectants of BJAB cells expressing green fluorescence protein (GFP)-CD20 were generated by electroporation at 250 V and 500 microfarads (Gene Pulser II; Bio-Rad) with 30 μg of DNA. GFP was fused to the amino terminus of CD20 by inserting human CD20 cDNA into the cloning site of the pEGFP expression vector (Clontech) using XhoI/SacII. CD20-positive cells were sorted by flow cytometry (FACStar cytometer; BD Biosciences) and maintained with Geneticin (Invitrogen) at 1 mg/ml.Antibodies and Reagents—Monoclonal antibodies 2H7 (IgG2b anti-CD20) and 9.4 (IgG2b anti-CD45) were provided by Dr. J. Ledbetter (Seattle, WA), and the isotype switch variants of NK1.B20 were provided by Dr. Anne-Marie Hekman (Netherlands Cancer Institute, Amsterdam) (13Hooijberg E. Sein J.J. van den Berk P.C. Hekman A. Hybridoma. 1996; 15: 23-31Crossref PubMed Scopus (10) Google Scholar). B1 (IgG2a anti-CD20) was purchased from Coulter (Hialeah, FL). Anti-CD59 antibody (MEM43/5) was purchased from Sanbio (Uden, The Netherlands). 2H7 and B1 were conjugated to Alexa 488 (A488) using the Alexa Fluor 488 protein labeling kit (Molecular Probes, Inc., Eugene, OR). Goat anti-rabbit IgG conjugated to Cy3 was purchased from Jackson ImmunoResearch. Cholera toxin subunit B (CTB) and rabbit anti-CTB were purchased from Sigma. For immunoblotting, anti-Gαi antibody was purchased from Oncogene (Boston, MA), anti-Lyn from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), anti-actin from Roche Applied Science, and anti-CD45 antibody from Transduction Laboratories (Lexington, KY). Rabbit antiserum directed against a CD20 cytoplasmic region peptide was generated as described (14Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (71) Google Scholar). Horseradish peroxidase conjugates of Protein A, rabbit anti-mouse IgG, and CTB were purchased from Bio-Rad, Southern Biotechnology Associates (Birmingham, AL), and Sigma, respectively.Fab fragments of 2H7 were produced using the ImmunoPure Fab preparation kit (Pierce). Purity was assessed by SDS-PAGE separation of titrated 2H7 mAb, both intact and digested, followed by Western blot using biotin-conjugated anti-mouse IgG2b detected with avidin-horseradish peroxidase (both reagents from Southern Biotechnology Associates). The limit of detection of intact 2H7 mAb was 1 ng. The initial Fab preparation was contaminated with ∼1 ng of intact mAb/μg of protein; however, after two additional clearance steps with protein A-Sepharose, there was no detectable intact mAb in 5 μg of Fab protein.Triton X-100 and CHAPS were purchased from Pierce. Thesit, Brij 35, Brij 58, and Brij 96 were purchased from Sigma. Glutaraldehyde and osmium tetroxide were from Electron Microscopy Sciences (Fort Washington, PA) and paraformaldehyde (PFA) from BDH Chemicals.Rafts Isolations and Immunoblots—Cell lysis in detergents (all used at 1%, except Triton X-100, which was also used at 0.05%) and sucrose density gradient centrifugation were done with slight modifications of the protocol previously described (8Deans J.P. Robbins S.M. Polyak M.J. Savage J.A. J. Biol. Chem. 1998; 273: 344-348Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, 108 cells were washed and then lysed for 15 min in ice-cold lysis buffer of detergent in MBS (25 mm MES, 150 mm NaCl) containing enzyme inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm NaVO4, 1 mm NaMoO4, 1 mm phenylmethylsulfonyl fluoride, 1 mm EDTA). Lysates were mixed with an equal volume of 80% sucrose in MBS plus inhibitors, overlayered with 5 ml of 30% and 5 ml of 5% sucrose in MBS plus inhibitors, and then centrifuged at 235,000 × g for 17 h. From the top of the gradients, eight 1.5-ml fractions were collected on ice. Sample preparation for CD20 translocation experiments was as described (8Deans J.P. Robbins S.M. Polyak M.J. Savage J.A. J. Biol. Chem. 1998; 273: 344-348Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, after incubation with antibodies, the cells were lysed in 1% Triton X-100 and separated into soluble and insoluble fractions by centrifugation for 15 min at 14,000 × g.For GM1 dot blots, nitrocellulose membranes (Schleicher & Schuell) were spotted with equal cell equivalents from each gradient fraction. For immunoblots, equal cell equivalents from each gradient fraction were mixed with SDS sample buffer, heated to 95 °C for 5 min, separated by SDS-PAGE under reducing conditions, and transferred to Immobilon P (Millipore Corp., Bedford, MA). All membranes were blocked with 5% bovine serum albumin. CTB-horseradish peroxidase (Sigma) was used to detect GM1. Other blots were performed as described (8Deans J.P. Robbins S.M. Polyak M.J. Savage J.A. J. Biol. Chem. 1998; 273: 344-348Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). All blots were developed using SuperSignal chemiluminescent substrate (Pierce) and visualized using the Fluor-S Max (Bio-Rad) imaging system. Quantity One software was used to quantitate the signal.Flow Cytometry—Cells were incubated with anti-CD20 mAb, followed by fluorescein isothiocyanate-conjugated anti-mouse IgG. The data were acquired using a Becton Dickinson FACScan (BD Biosciences) and analyzed using the FlowJo program (Tree Star, Inc., San Carlos, CA).Immunofluorescence Imaging—Cells were fixed in 1% PFA at room temperature for 5 min and then incubated with antibody as indicated or incubated with antibody prior to fixation. Fixed and unfixed PBMC (106) were incubated with 1 μg of 2H7-A488 in 100 μl of PBS at 37 °C for 15 min. Fixed Ramos B cells were incubated with B1- or 2H7-A488 (1 μg/106 cells) for 30 min at 37 °C; unfixed Ramos cells were incubated with the same antibodies for 10 min at 37 °C, washed, and fixed. For colocalization studies, 106 BJAB (GFP-CD20) cells were fixed in 1% PFA; incubated with anti-CD59, anti-CD20, or anti-CD45; washed; and further incubated with anti-mouse IgG-Cy3.Fluorescence imaging was done with a Leica DM RXA microscope attached to a 14-bit cooled CCD camera (Princeton Instruments, Monmouth Junction, NJ). Digital deconvolution was performed using the MicroTome software (VayTek, Fairfield, IA). In some experiments, imaging of PBMC was with DeltaVision Image Restoration Microscopy System (Applied Precision, Issaquah, WA).Electron Microscopy—For transmission EM, Ramos cells (1 × 107) were incubated with mouse IgG anti-CD20 (15 μg in 500 μl of PBS), washed in PBS, and then incubated in colloidal gold (6 nm)-conjugated goat anti-mouse IgG (Electron Microscopy Sciences). After washing, the cells were fixed in cold 2.5% glutaraldehyde in PBS with rotation at 4 °C for 1 h. After washing three times (20 min each) with the same buffer, the cells were pelleted and embedded in 3% agar. The agarized pellet was cut into 1-mm slices and immersed in 1% osmium tetroxide in PBS for 1 h at room temperature. The pellets were rinsed in distilled water, dehydrated in graded ethanol series, and embedded in Epon 812. Thin sections were cut with a diamond knife, mounted on naked copper grids, and viewed with a Hitachi (Schaumburg, IL) H-7000 transmission electron microscope.For scanning EM, Ramos cells (1 × 107) were fixed in cold 1% glutaraldehyde/PBS with rotation at 4 °C for 1 h, washed three times in PBS, and dehydrated in graded ethanol series. The cell pellet was resuspended in 10 μl of 100% ethanol and transferred to a poly-l-lysine-coated coverslip for critical point drying. Coverslips were then sputter-coated with gold palladium, dried, and mounted on the sample stage of a Philips XL30 scanning electron microscope (FEI Co., Hillsboro, OR).RESULTSCD20 Is Constitutively Associated with Membrane Rafts—To test whether the use of detergents other than Triton X-100 might reveal a significant presence of CD20 in rafts in the absence of antibody engagement, Ramos B cells were lysed in the series of detergents indicated in Fig. 1. After sucrose density gradient centrifugation, fractions were tested by immunoblot for the presence of CD20. As controls, the gradient fractions were also blotted for actin, raft markers GM1 and Gαi, and nonraft marker CD45. CD20 was solubilized by Triton X-100 at 1%, as expected. However, in 0.05% Triton and in all other detergents except Brij 96, 30–80% of CD20 redistributed to the buoyant raft fractions 3–5. Glycosphinglipid GM1 localized to the raft fractions in all detergents. The acylated heteromeric G protein subunit Gαi was found predominantly in the raft fractions in all detergents except Brij 96. The distribution of actin was similar in all gradients. CD45, an abundant nonraft protein, was not detected in the raft fractions in any detergent, indicating selectivity of the isolations.Brij 58 and Brij 35 extracted the most CD20 into raft fractions. To further assess the distribution of plasma membrane-associated proteins in Brij 58 gradients, Ramos or BJAB B cells were surface-labeled with biotin before lysis, and fractions from the gradients were probed by Western blot with avidin-horseradish peroxidase. Results from BJAB cells are shown in Fig. 2; similar data were obtained with Ramos. Proteins found in raft fractions 3–5 appeared to include a distinct subset and represented ∼30% of all surface-labeled proteins, as estimated by densitometry analysis (Fig. 2, A, left panel, and B). After pretreatment with methyl-β-cyclodextrin (MBC), <13% of surface-labeled proteins were detected in fractions 3–5 (Fig. 2, A, right panel and B), demonstrating the cholesterol dependence expected of most protein-raft associations. The buoyancy of both CD20 and Gαi on Brij 58 gradients was sensitive to cellular pretreatment with MBC and was restored by subsequent incubation with cholesterol-loaded MBC (Fig. 2C). Interestingly, the buoyancy of GM1 was less dependent on cholesterol. Quantitative analysis showed that the amount of GM1 in rafts (fractions 3–5) was reduced less than 20% by MBC treatment compared with more than 95 and 90% reduction for CD20 and Gαi, respectively.Fig. 2Cholesterol dependence of CD20-raft association. A, BJAB cells were surface-labeled with biotin and incubated in the absence or presence of 2% MBC for 30 min before lysis in Brij 58. Lysates were fractionated by sucrose density gradient centrifugation, and fractions were analyzed by blotting with avidin-horseradish peroxidase. Similar data were obtained using Ramos cells. B, the percentage of biotinylated proteins in each fraction was estimated using Quantity One software. Data are the average ± S.E. from three experiments. C, cells were incubated in the absence or presence of MBC. Samples after MBC treatment were followed by further incubation with 2% cholesterol-loaded MBC for 30 min, as indicated. The cells were then lysed in Brij 58 and fractionated on sucrose density gradients. Fractions were immunoblotted for CD20, Gαi, and GM1 as indicated. Data shown are representative of two experiments.View Large Image Figure ViewerDownload (PPT)Cell Surface Distribution of CD20 and Colocalization with Raft Marker CD59 —Since constitutive raft association of CD20 was shown in the above experiments, we examined its cell surface distribution before and after ligation with mAb 2H7, which induces 95% of CD20 protein to become insoluble in 1% Triton X-100 (8Deans J.P. Robbins S.M. Polyak M.J. Savage J.A. J. Biol. Chem. 1998; 273: 344-348Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 12Polyak M.J. Tailor S.H. Deans J.P. J. Immunol. 1998; 161: 3242-3248PubMed Google Scholar). For these experiments, we used peripheral blood B cells (Fig. 3A), Ramos cells (Fig. 3, B and C), and BJAB cells transfected with a GFP-CD20 construct (Fig. 3, E and F), and results obtained with all three B cell sources were concordant. The pattern of CD20 staining on cells that were fixed prior to labeling was similar to that observed when cells were fixed after incubation with the 2H7 mAb (Fig. 3A). There was no apparent large scale change in the distribution of CD20 after antibody binding. CD20 was not evenly distributed around the membrane but was somewhat punctate. Similar data were obtained when cells were fixed with higher concentrations of PFA and/or in the presence of glutaraldehyde (data not shown) and also when CD20 was labeled with fluorochrome-conjugated highly purified Fab fragments of 2H7 to exclude cross-linking effects (Fig. 3B). Cell surface distribution of CD20 was not different when CD20 was ligated and labeled with B1 mAb, which induces minimal ( 90%) were associated with membrane protrusions. A representative image is shown in Fig. 4; low magnification of transmission and scanning EM images of Ramos B cells in the lower panels of Fig. 4 illustrate the gross plasma membrane architecture of these cells, which is similar to that reported previously for primary B cells (16Perkins W.D. Karnovsky M.J. Unanue E.R. J. Exp. Med. 1972; 135: 267-276Crossref PubMed Scopus (53) Google Scholar, 17Gonatas N.K. Antoine J.C. Stieber A. Avrameas S. Lab. Invest. 1972; 26: 253-261PubMed Google Scholar, 18Lin P.S. Cooper A.G. Wortis H.H. N. Engl. J. Med. 1973; 289: 548-551Crossref PubMed Scopus (97) Google Scholar, 19Polliack A. Lampen N. Clarkson B.D. De Harven E. Bentwich Z. Siegal F.P. Kunkel H.G. J. Exp. Med. 1973; 138: 607-624Crossref PubMed Scopus (241) Google Scholar). No gold particles were observed in isotype control samples (data not shown).Fig. 4CD20 is localized to microvilli. Ramos cells were labeled with 2H7 anti-CD20 or isotype control and gold (6 nm)-conjugated goat anti-mouse IgG and then prepared for transmission EM (upper image; scale bar, 200 nm). Similar results were obtained after labeling with B1 anti-CD20, and no gold particles were observed in the isotype controls (data not shown). A low magnification transmission EM image is shown in the lower left (scale bar, 2 μm), and a scanning EM image is shown in the lower right (scale bar, 2 μm).View Large Image Figure ViewerDownload (PPT)Antibody-induced Insolubility in 1% Triton—Although unligated CD20 is soluble in 1% Triton, antibody binding can induce almost complete insolubility of CD20 in this detergent (8Deans J.P. Robbins S.M. Polyak M.J. Savage J.A. J. Biol. Chem. 1998; 273: 344-348Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) (see Fig. 5A). Antibody-ligated Triton-insoluble CD20 was found in the low density region of sucrose gradients and not in the high density pellet (Fig. 5A, fraction P). The buoyancy of ligated Triton-insoluble CD20 was sensitive to cholesterol depletion and restored by subsequent incubation with cholesterol-loaded MBC (Fig. 5A). The distribution of the Src-family kinase Lyn on these gradients is shown for comparison. In light of the constitutive CD20-raft association demonstrated earlier, these data suggest that induced Triton insolubility reflects increased strength of the association with rafts. We then investigated the mechanism underlying this effect using a rapid and sensitive procedure, described previously (12Polyak M.J. Tailor S.H. Deans J.P. J. Immunol. 1998; 161: 3242-3248PubMed Google Scholar), in which the loss of CD20 from the soluble fraction of Triton lysates caused by antibody binding is monitored as well as the corresponding gain of CD20 in the insoluble material obtained after microcentrifugation. To confirm the cholesterol dependence of CD20 insolubility using this procedure, Ramos B cells were pretreated with MBC before the addition of 2H7 anti-CD20 mAb. Both soluble and insoluble fractions were collected and probed for the presence of CD20 (Fig. 5B). As expected, antibody-induced loss of CD20 from the soluble fractions (lane 5) was prevented by preincubation of the cells with MBC (lanes 6–8). The pellet fractions showed the reverse results (i.e. 2H7-induced CD20 Triton-insolubility was prevented by MBC pretreatment).Fig. 5Antibody-induced insolubility of CD20 in 1% Triton. A, untreated, MBC, or cholesterol/MBC-treated Ramos cells were incubated with isotype control or 2H7 mAb before lysis in 1% Triton X-100, fractionated on sucrose density gradients, and immunoblotted for CD20 or Lyn. B, Ramos cells were pretreated with MBC for the times indicated, and then 2H7 anti-CD20 was added for an additional 15 min before lysis in 1% Triton X-100. The soluble and insoluble fractions were collected and probed by immunoblot for CD20. Results are representative of three experiments in A and two experiments in B. For these and similar experiments shown in later figures, equal loading was demonstrated by actin blot or Coomassie Blue staining, but it is not shown for clarity of presentation.View Large Image Figure ViewerDownload (PPT)Antibody-induced association of CD20 with 1% Triton-resistant rafts could potentially be mediated by the actin cytoskeleton, by signaling events or post-translational modifications, by clustering on a scale that is not obvious at the level of light microscopy, or by a CD20-intrinsic mechanism such as a change in conformation. Cytoskeleton-associated proteins are among the most abundant proteins in membrane rafts (20von Haller P.D. Donohoe S. Goodlett D.R. Aebersold R. Watts J.D. Proteomics. 2001; 1: 1010-1021Crossref PubMed Google Scholar). The membrane skeleton has also been suggested to regulate the size of lipid rafts (21Kusumi A. Sako Y. Curr. Opin. Cell Biol. 1996; 8: 566-574Crossref PubMed Scopus (316) Google Scholar). As shown in Fig. 6A, pretreating Ramos cells with 1 μm cytochalasin D for up to 16 h did not affect CD20 Triton insolubility induced b
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