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• W1995030412 abstract "Recent studies have highlighted the existence of discrete microdomains at the cell surface that are distinct from caveolae. The function of these microdomains remains unknown. However, recent evidence suggests that they may participate in a subset of transmembrane signaling events. In hematopoietic cells, these low density Triton-insoluble (LDTI) microdomains (also called caveolae-related domains) are dramatically enriched in signaling molecules, such as cell surface receptors (CD4 and CD55), Src family tyrosine kinases (Lyn, Lck, Hck, and Fyn), heterotrimeric G proteins, and gangliosides (GM1 and GM3). Human T lymphocytes have become a well established model system for studying the process of phorbol ester-induced down-regulation of CD4. Here, we present evidence that phorbol 12-myristate 13-acetate (PMA)-induced down-regulation of the cell surface pool of CD4 occurs within the LDTI microdomains of T cells. Localization of CD4 in LDTI microdomains was confirmed by immunoelectron microscopy. PMA-induced disruption of the CD4-Lck complex was rapid (within 5 min), and this disruption occurred within LDTI microdomains. Because PMA is an activator of protein kinase C (PKC), we next evaluated the possible roles of different PKC isoforms in this process. Our results indicate that PMA induced the rapid translocation of cytosolic PKCs to LDTI microdomains. We identified PKCα as the major isoform involved in this translocation event. Taken together, our results support the hypothesis that LDTI microdomains represent a functionally important plasma membrane compartment in T cells. Recent studies have highlighted the existence of discrete microdomains at the cell surface that are distinct from caveolae. The function of these microdomains remains unknown. However, recent evidence suggests that they may participate in a subset of transmembrane signaling events. In hematopoietic cells, these low density Triton-insoluble (LDTI) microdomains (also called caveolae-related domains) are dramatically enriched in signaling molecules, such as cell surface receptors (CD4 and CD55), Src family tyrosine kinases (Lyn, Lck, Hck, and Fyn), heterotrimeric G proteins, and gangliosides (GM1 and GM3). Human T lymphocytes have become a well established model system for studying the process of phorbol ester-induced down-regulation of CD4. Here, we present evidence that phorbol 12-myristate 13-acetate (PMA)-induced down-regulation of the cell surface pool of CD4 occurs within the LDTI microdomains of T cells. Localization of CD4 in LDTI microdomains was confirmed by immunoelectron microscopy. PMA-induced disruption of the CD4-Lck complex was rapid (within 5 min), and this disruption occurred within LDTI microdomains. Because PMA is an activator of protein kinase C (PKC), we next evaluated the possible roles of different PKC isoforms in this process. Our results indicate that PMA induced the rapid translocation of cytosolic PKCs to LDTI microdomains. We identified PKCα as the major isoform involved in this translocation event. Taken together, our results support the hypothesis that LDTI microdomains represent a functionally important plasma membrane compartment in T cells. Recent studies have highlighted that the plasma membrane is not homogenous but instead consists of a variety of discrete microdomains (1Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar, 2Parolini I. Sargiacomo M. Lisanti M.P. Peschle C. Blood. 1996; 87: 3783-3794Crossref PubMed Google Scholar, 3Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar, 4Sorice M. Parolini I. Sansolini T. Garofalo T. Dolo V. Sargiacomo M. Tai T. Peschle C. Torrisi M.R. Pavan A. J. Lipid Res. 1997; 38: 969-980Abstract Full Text PDF PubMed Google Scholar). CD4 is an ∼ 55–59-kDa membrane glycoprotein expressed on the surface of T helper cells and to a lesser extent on monocytes/macrophages. It is the human receptor for HIV, 1The abbreviations used are: HIV, human immunodeficiency virus; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; LDTI, low density Triton-insoluble; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; FITC, fluorescein isothiocyanate whose binding allows the entrance of HIV to target cells (5Dalgleish A.G. Beverly P.C.L. Clapham P.R. Crawford D.H. Greaves M.F. Weiss R.A. Nature. 1984; 312: 763-767Crossref PubMed Scopus (2585) Google Scholar). CD4 is considered to be the TCR co-receptor in T-cell activation and thymic selection (6Blue M.L. Hafler D.A. Craig K.A. Levine H. Schlossman S.F. J. Immunol. 1987; 139: 3949-3954PubMed Google Scholar). In this regard, it binds to major histocompatibility complex class II epitopes, thereby strengthening cell-to-cell contact and TCR-major histocompatibility complex formation (7Gay D. Buus S. Pasternak J. Kappler J. Marrack P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5629-5634Crossref PubMed Scopus (52) Google Scholar). In addition, CD4 itself mediates intracellular signals that influence TCR-CD3 complex formation and augments the cellular response (8Veillette A. Bookman M.A. Horak E.M. Samelson L.E. Bolen J.B. Nature. 1989; 338: 257-259Crossref PubMed Scopus (530) Google Scholar). It is well established that this effect is due to CD4 interaction with Lck (9Rudd C.E. Trevillyan J.M. Dasgupta J.D. Wong L.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (616) Google Scholar), a member of the Src family of tyrosine kinases. Lck is anchored to the cytoplasmic side of the membrane via lipid modifications and is involved in CD3 γ, ε chain phosphorylation (10Glaichenhaus N. Shastri N. Littman D.R. Turner J.M. Cell. 1991; 64: 511-520Abstract Full Text PDF PubMed Scopus (349) Google Scholar). Lck also plays a role in regulating the endocytic properties of CD4, thereby controlling the cellular distribution of this co-receptor (11Pelchen-Matthews A. Boulet I. Littman D.R. Fagard R. Marsh M. J. Cell Biol. 1992; 117: 279-290Crossref PubMed Scopus (123) Google Scholar). Treatment of T cells with phorbol esters such as PMA is one of the methods used to mimic modulation of CD4 that occurs during the antigen encounter (12Acres B.R. Conlon P.J. Mochizuki D.Y. Gallis B. J. Biol. Chem. 1986; 261: 16210-16214Abstract Full Text PDF PubMed Google Scholar). Upon activation, cytoplasmatic serine residues of CD4 are phosphorylated most likely via an isoform or isoforms of PKC (13Veillette A. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 55: 301-308Abstract Full Text PDF PubMed Scopus (1133) Google Scholar,14Alexander D.R. Cantrell D.A. Immunol. Today. 1989; 10: 200-205Abstract Full Text PDF PubMed Scopus (100) Google Scholar). PKCs are a family of at least 12 isoenzymes, whose 8 isotypes (α, β1, β2, δ, ε, ζ, η, and θ) are expressed in T cells (15Lucas S. Marais R. Graves J.D. Alexander D. Parker P.J. Cantrell D.A. FEBS Lett. 1990; 260: 53-56Crossref PubMed Scopus (33) Google Scholar, 16Mischak H. Kolch W. Goodnight J. Davidson W.F. Rapp U. Rose-John S. Mushinsky J.F. J. Immunol. 1991; 147: 3981-3987PubMed Google Scholar) and are responsible for CD4 down-modulation by endocytosis through clathrin-coated pits (17Pelchen-Matthews A. Parson I.J. Marsh M. J. Exp. Med. 1993; 178: 1209-1222Crossref PubMed Scopus (143) Google Scholar). In this regard, CD4 lacking intracellular serine residues (a possible target for PKC phosphorylation) is not down-regulated by phorbol esters (18Shin J. Doyle C. Yang Z. Kappes D. Strominger J.L. EMBO J. 1990; 9: 425-434Crossref PubMed Scopus (87) Google Scholar, 19Doyle C. Shin J. Dunbrack R.L. Strominger J.L. Immunol. Rev. 1989; 109: 17-37Crossref PubMed Scopus (11) Google Scholar). CD4 expression returns to normal only upon prolonged PMA stimulation, which exhausts cytoplasmic stores of PKC (20Hoxie J.A. Matthews D.M. Callahan K.J. Cassel D.L. Cooper R.A. J. Immunol. 1986; 137: 1194-1201PubMed Google Scholar). PKCs differ in substrate specificity, cofactor requirements, tissue and cellular distributions, subcellular localizations (21Hoessli D.C. Rungger-Brandle E. Curr. Opin. Cell Biol. 1996; 8: 168-173Crossref PubMed Scopus (407) Google Scholar), and regulatory mechanisms (22Stabel S. Parker P.J. Pharmacol. Ther. 1991; 51: 71-95Crossref PubMed Scopus (454) Google Scholar) that lead to their differential translocation in the cell following stimulation (23Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1218) Google Scholar). In T lymphocytes, PMA activation induces PKCα, β1, and β2 redistribution from a diffusely cytoplasmic localization to a discrete focal distribution around the plasma membrane and nucleus (24Berry N. Ase K. Kikkawa U. Kishimoto A. Nishizuka Y. J. Immunol. 1989; 143: 1407-1413PubMed Google Scholar). Little is known about the specific role played by each of the multiple isoforms present in a given cell type, although the involvement of PKCθ in T cell activation following stimulation by antigen presenting cells has been recently described. In this regard PKCθ was spatially restricted to the site of contact, where receptors on the T cells encounter their counterparts on antigen presenting cells (25Monks C.R.F. Kupfer H. Tamir I. Barlow A. Kupfer A. Nature. 1997; 385: 83-86Crossref PubMed Scopus (494) Google Scholar). The subcellular localization of a given PKC isoform may represent an important clue in determining the specific function of a given PKC isoform. Electron microscopy and plasma membrane fractionation in the absence of detergent have demonstrated that the PKCα isoform is enriched with caveolae (26Smart E.J. Ying Y.S. Anderson R.G.W. J. Cell Biol. 1995; 131: 929-938Crossref PubMed Scopus (159) Google Scholar, 27Smart E.J. Ying YS Mineo C. Anderson R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar). The co-existence in the same cell of caveolae and membrane “rafts” enriched in glycolipids has been described (28Parton R.G. Simons K. Science. 1995; 269: 1398-1399Crossref PubMed Scopus (296) Google Scholar, 29Liu J. Oh P. Horner T. Rogers R.A. Schnitzer J. J. Biol. Chem. 1997; 272: 7211-7222Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 30Harder T. Simons K. Curr. Opin. Cell Biol. 1997; 9: 534-542Crossref PubMed Scopus (718) Google Scholar) as well as membrane rafts in cells devoid of morphologically recognizable caveolae such as neuronal and hematopoietic cells (2Parolini I. Sargiacomo M. Lisanti M.P. Peschle C. Blood. 1996; 87: 3783-3794Crossref PubMed Google Scholar, 3Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar, 31Wu C. Butz S. Ying Y. Anderson R.G.W. J. Biol. Chem. 1997; 272: 3554-3559Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 32Engelman J.A. Wykoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. J. Biol. Chem. 1997; 272: 16374-16381Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). These rafts have also been termed LDTI or “caveolae-related domains.” We recently purified and characterized these low density Triton-insoluble (LDTI) microdomains from hematopoietic cells. These LDTI microdomains morphologically and biochemically resemble raft domains and were highly enriched in signal transducing molecules such as a subset of cell surface receptors, Src family tyrosine kinases, heterotrimeric G proteins, and gangliosides (GM3) (2Parolini I. Sargiacomo M. Lisanti M.P. Peschle C. Blood. 1996; 87: 3783-3794Crossref PubMed Google Scholar,4Sorice M. Parolini I. Sansolini T. Garofalo T. Dolo V. Sargiacomo M. Tai T. Peschle C. Torrisi M.R. Pavan A. J. Lipid Res. 1997; 38: 969-980Abstract Full Text PDF PubMed Google Scholar). New insights into the dynamic clustering of raft domains have highlighted their potential role as a starting point for many membrane-linked processes, including certain transmembrane signaling events (30Harder T. Simons K. Curr. Opin. Cell Biol. 1997; 9: 534-542Crossref PubMed Scopus (718) Google Scholar). However, in the hematopoietic system, the exact function of these domains has not yet been established. To address this issue, we have analyzed the well described process of CD4 internalization in human T cells that is induced by activation with PMA. Here, we show that this process takes place within LDTI microdomains. More specifically, we demonstrate that upon PMA treatment (i) the CD4-Lck complex is disrupted within LDTI domains; (ii) CD4 shifts from LDTI domains to a Triton-soluble particulate fraction in a time-dependent manner, whereas Lck remains within the LDTI domain; and (iii) many PKC isoforms are activated and translocated from the cytosol to LDTI domains but at different rates. In addition, PKCα appears to be the most abundant isoform within LDTI domains, suggesting that it plays an important role in this process. Anti-CD4 monoclonal antibody used for Western blotting was purchased from Novocastra (Newcastle-upon-Tyne, UK). Anti-CD4 monoclonal antibody used for immunoprecipitation was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). For FACS analysis and immunoelectron microscopy anti-CD4, OKT4 was purchased from Ortho Diagnostic System (Raritan, NJ). Anti-Lck, anti-PKCβ1, β2, δ, ε, and η polyclonal antibodies were purchased from Santa Cruz Biotechnologies; anti-Fyn, anti-PKCα, and anti-PKCθ monoclonal antibodies were purchased from Transduction Laboratories, Inc. (Lexington, KY); anti-PKCζ was purchased from Calbiochem (La Jolla, CA). Anti-monoclonal or -polyclonal secondary antibodies horseradish peroxidase-conjugated were purchased from Bio-Rad. Biotin-NHS was purchased from Calbiochem, and streptavidin-horseradish peroxidase conjugated was from Pierce. Gö 6976 and Gö 6850 were obtained from Calbiochem. PKCα purified enzyme was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). PMA and Histone H1, Type IIIS, were obtained from Sigma. Anti-Gi antibody was a generous gift of Dr. Tommaso Costa. An established protocol was followed to prepare total cell membranes (or particulate), enriched in plasma membrane, from human peripheral lymphocytes, with some modifications (65Lehel C. Olah Z. Mischak H. Mushinski J.F. Anderson W.B. J. Biol. Chem. 1994; 269: 4761-4766Abstract Full Text PDF PubMed Google Scholar). Briefly, 1 × 109 lymphocytes were surface labeled by incubation with biotin-NHS (0.5 mg/ml) for 30 min at 4 °C. After washing with ice-cold serum-free Dulbecco's modified Eagle's medium and then with PBS, cells were incubated with PMA (100 ng/ml) at 37 °C for the indicated times in warm RPMI medium. After three washes with ice-cold PBS, cells were Dounce-homogenized with 2 ml of lysis buffer (20 mm Tris, pH 8,0, 2 mm EGTA containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin A). The homogenate was first centrifuged at 1,000 × g for 5 min at 4 °C, and then the supernatant was centrifuged at 2,000 ×g for 5 min to remove nuclear debris. The resulting supernatant was centrifuged at 60,000 rpm for 30 min to produce a nucleus-free membrane fraction (pellet) and a cytosol fraction (supernatant). Proteins were quantified by Peterson method (33Peterson G.L. Methods Enzymol. 1983; 91: 95-119Crossref PubMed Scopus (1142) Google Scholar) and then resolved by SDS-PAGE, transferred to a 0.22-μm nitrocellulose filter (Amersham Life Science, Buckinghamshire, UK), and blocked with 4% nonfat milk and 1% BSA in TBST (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0,05% Tween 20). The membrane was then probed with streptavidin-horseradish peroxidase conjugated at 1:30,000 dilution in TBST for 1 h at room temperature. The membrane was washed six times with TBST and incubated with SuperSignal chemiluminescence ULTRA (Pierce) according to the manufacturer's instructions. Reactive proteins were detected by autoradiography on Kodak T-Mat G/RA film (Eastman Kodak, Rochester, NY). LDTI complexes were isolated as described previously (2Parolini I. Sargiacomo M. Lisanti M.P. Peschle C. Blood. 1996; 87: 3783-3794Crossref PubMed Google Scholar, 34Sargiacomo M. Sudol M. Tang Z.L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar). Briefly, 1 × 109 lymphocytes were washed and lysed in 1 ml of MBS (25 mm MES, pH 6, 5, 150 mm NaCl) or were treated as described to recover membrane fractions (M). Next both preparations were Dounce homogenized in buffer containing 0, 0.02, 0.1, or 1% Triton X-100 and 0.1 mg/ml phenylmethylsulfonyl fluoride, adjusted to 40% sucrose, and placed at the bottom of four different ultracentrifuge tubes. A 5–30% linear sucrose gradient was then placed above the lysate, and the mixture was centrifuged at 45,000 RPM, 16 h, 4 °C in a SW60 rotor (Beckman Instruments, Palo Alto, CA). In both cases LDTI, visible as a band migrating at approximately 20% sucrose, was harvested and washed twice with MBS at 14,000 RPM for 30 min at 4 °C and then protein quantitated. 1 × 109 lymphocytes consisting of 50 mg of total protein, yielded a mean of 7 mg of particulate fractions, and 110 μg of LDTI fractions. Thus LDTI represents 0.2% of the initial homogenate and 1.5% of membrane fraction, whereas membrane represents 14% of the initial homogenate. LDTI, total cell lysates or total membrane proteins were resolved by 8% SDS-PAGE under reducing conditions and transferred to nitrocellulose filter. The blots were blocked using 5% nonfat milk in TBST for 1 h at room temperature, followed by incubation with anti-Lck polyclonal (dilution 1:100), anti-Fyn monoclonal (dilution 1:400), anti-Gi polyclonal (dilution 1:2000), anti CD4 monoclonal (dilution 1:200) or anti-PKC-specific antibodies in TBST for 1 h at room temperature. PKCα was 1:4000 diluted, PKCθ was 1:250 diluted, and the other PKC isoforms were 1:100 diluted. After washing with TBST, each filter was incubated with the appropriate secondary antibody-horseradish peroxidase conjugated at 1:3000 dilution for 1 h at room temperature. Reactive proteins were detected as described above. For immunoprecipitation experiments LDTI domains or membrane fraction from biotin-labeled lymphocytes were prepared as described above. 1–5 μg of protein were precleared with 30 μl of a 50% slurry protein A/G-agarose (Pierce) and 1 μg of nonimmune serum in 0.5 ml of lysis buffer (150 mm NaCl, 10 mmTris-HCl, pH 7.4, 1% Nonidet P-40, 10% glycerol, 0.1 mg/ml phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin A) for 1 h at 4 °C. CD4 antibody (0.5 μg/ml) or PKCα (1 μg/ml) or PKCδ (1 μg/ml) were then added to the sample and kept overnight at 4 °C, followed by incubation with prewashed beads (40 μl), 1 h at 4 °C. The beads were spun down and washed four times with lysis buffer, resuspended in 30 μl of SDS-PAGE sample buffer under reducing conditions, boiled, and spun down. The supernatant was loaded on a 8% SDS-PAGE. Cell surface CD4 was detected by blotting the filter with streptavidin-horseradish peroxidase conjugated under the conditions described for detection of surface proteins. Using this procedure only one band corresponding to 55-kDa CD4 was detectable. 1 μg of LDTI complexes isolated from PMA-treated cells were resuspended in 20 μl of kinase reaction buffer (20 mm Hepes, pH 7.4, 5 mm MgCl2, and 1 mm MnCl2) supplemented with Gö 6976 (10−7m) and 5 μCi of [γ-32P]ATP for 10 min at room temperature. The reaction was stopped by addition of 20 μl of Laemmli sample buffer (2×) under reducing conditions. The mixture was boiled, separated on 10% SDS-PAGE that was then dried, and exposed to Kodak XAR film. In other experiments, to test the activity of PKCα, 5 μg of Histone type IIIS and 25 ng of PKCα purified enzyme were incubated with 10 μg of phospatidylserine in ADB buffer (1 mm sodium ortovanadate, 25 mm β-glycerophosphate, 20 mm MOPS, pH 7.2, 1 mm dithiothreitol, 1 mm CaCl2, 5 mm MgCl2), prior to the addition of 5 μCi of [γ-32P]ATP. CD4+ lymphocytes (1 × 106 in 1 ml of PBS) were incubated with PMA as reported above. PMA-treated and untreated cells were then fixed with acetone/methanol 1:1 (v/v) for 10 min at 4 °C. Cells were soaked in Hank's balanced salt solution for 30 min at 25 °C and incubated for 20 min at 25 °C in the blocking buffer of 2% BSA in PBS containing 5% glycerol and 0.2% Tween 20. Cells were then labeled with anti-PKCα, δ, or ε polyclonal antibodies (Santa Cruz Biotechnology) for 1 h at 4 °C. After three washes in PBS, cells were incubated with FITC-conjugated goat anti-rabbit IgG (Sigma) for 30 min at 4 °C. After washing three times in PBS, pH 7.4, cells were then incubated for 1 h at 4 °C with anti-Lck monoclonal antibody, followed by 3 washes in PBS and the addition (30 min at 4 °C) of goat anti-mouse IgG (γ-chain-specific) conjugated with Texas Red (Calbiochem Biochem). Cells were finally washed three times in PBS and then mounted upside down onto a glass slide in 5 ml of glycerol/Tris-HCl, pH 9.2. The coverslips were sealed with nail varnish to prevent evaporation and stored at 4 °C before imaging. The images were acquired through a confocal laser scanning microscope (Sarastro 2000, Molecular Dynamics) equipped with a NIKON OPTIPHOT microscope (objective 60/1.4 oil) and an Argon Ion Laser (25 mW output). Simultaneously, the green (FITC) and the red (Texas Red, which reduces greatly overlapping) fluorophores were excited at 488 and 518 nm. Acquisition of single FITC-stained samples in dual fluorescence scanning configuration did not show contribution of green signal in red. Images were collected at 512 × 512 pixels (0.08 μm/pixel lateral dimension, 0.48 μm/pixel axial dimension). Serial optical sections were assembled in Depth-Coding (Molecular Dynamics) mode. Acquisition and processing were carried out using Image Space software (Molecular Dynamics). CD4 expression on untreated and PMA-treated lymphocytes was investigated using monoclonal antibody CD4 (OKT4) fluorescein conjugated (1:20 in PBS, 1% BSA for 30 min at 4 °C). After washing with PBS/BSA cells were fixed with 1% formaldehyde in PBS. Green fluorescence intensity was analyzed with FACS scan cytometer (Becton Dickinson). For every histogram 5000 cells were counted to evaluate the percentage of CD4+ cells. The percentage of surface expression at different times of incubation with PMA was calculated using the mean fluorescence divided by the mean fluorescence at time 0 minus the background fluorescence. To assess CD4 localization at the cell plasma membrane of Triton-treated cells we followed a previously reported protocol with some modifications (35Moldovan N.I. Heltianu C. Dimionescu N. Simionescu M. Exp. Cell Res. 1995; 219: 309-313Crossref PubMed Scopus (32) Google Scholar). Briefly, CD4+ cells were isolated from peripheral lymphocytes using the IsoCellTM human CD4 Isolation Kit (Pierce) according to the manufacturer's instruction. CD4+ cells were then incubated with monoclonal antibody (OKT4) at 1:5 dilution in 0.5 ml of PBS/BSA for 1 h at 4 °C, followed by two washes with ice-cold PBS. Cells were resuspended with 1 ml of paraformaldehyde 3% in PBS, pH 7.2 for 30 min at 4 °C. After washing, cells were left untreated or 1% Triton X-100-treated for 30 min at 4 °C, followed by a second incubation with monoclonal antibody (OKT4) for 1 h at 4 °C. After incubation with rabbit anti-mouse IgG (Sigma) (1:10 in PBS for 1 h at 4 °C), cells were fixed with glutaraldehyde (1% in PBS for 1 h at 4 °C), extensively washed, and then labeled with colloidal gold (18 nm, prepared by the citrate method) conjugated with protein A (Amersham Pharmacia Biotech) for 3 h at 4 °C. Control experiments were performed omitting the incubation with OKT4 monoclonal antibody in both untreated and 1% Triton X-100-treated lymphocytes. All samples were postfixed in 1% osmium tetroxide in Veronal acetate buffer, pH 7.4, for 2 h at 4 °C, stained with uranyl acetate (5 mg/ml), dehydrated in acetone, and embedded in Epon 812. Mophometric analysis of the length of plasma membrane in Triton X-100-treated cells and in untreated controls as well as quantification of colloidal gold granules were performed on 30 micrographs printed at the same magnification. The results were expressed as the means ± S.D. The detergent insolubility of caveolae and caveolae-related domains is based on their high content of cholesterol and sphingolipids; many distinct classes of lipid-modified signaling molecules are retained within these detergent-resistant membrane domains. However, certain caveolae- and caveolin-1-associated proteins are dissociated from these domains as a consequence of detergent treatment; these include receptor tyrosine kinases (such as epidermal growth factor receptor) and a variety of prenylated proteins (such as Ha-Ras) (36Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). In addition, a small amount of caveolin-1 is found in the Triton-soluble fraction, and this fraction of caveolin-1 is associated with the Golgi complex (37Kishore K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-634Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). More interestingly, recent evidence indicates that proteins can move in and out of caveolae-related domains, depending on their activations state and that this dynamic movement can be monitored by changes in Triton solubility and partitioning into low density Triton-insoluble domains (LDTI, a biochemically descriptive term for caveolae-related domains) (38Montixi C. Langlet C. Bernard A.M. Thimonier J. Dubois C. Wurbel M.A. Chauvin J.P. Pierres M. He H.T. EMBO J. 1998; 17: 5334-5348Crossref PubMed Scopus (560) Google Scholar). In a previous report, we demonstrated that CD4 is dramatically enriched within these LDTI domains in human lymphocytes (4Sorice M. Parolini I. Sansolini T. Garofalo T. Dolo V. Sargiacomo M. Tai T. Peschle C. Torrisi M.R. Pavan A. J. Lipid Res. 1997; 38: 969-980Abstract Full Text PDF PubMed Google Scholar). Here, we analyze the changes in CD4 localization after cell activation by phorbol esters (PMA). To study changes in CD4 localization following phorbol ester cell activation, we first analyzed the Triton sensitivity of CD4 in resting lymphocytes. Thus, we experimentally defined the optimal Triton X-100 concentration that is required for the isolation of Triton-insoluble CD4 from the bulk of soluble plasma membrane proteins (Fig.1 a). To this end, ∼ 1 × 109 lymphocytes were surface labeled with sulfo-NHS-biotin. Two mg of the cell membrane fraction (M) were prepared from these labeled lymphocytes and homogenized with increasing amounts of Triton X-100, followed by equilibrium density gradient centrifugation. Each tube was then divided into twelve 375-μl fractions, and 10 μl/fraction was subjected to SDS-PAGE and blotting with streptavidin-horseradish peroxidase. Only biotin-labeled cell surface proteins were detected by this procedure (Fig. 1 a,left).Figure 1Characterization and enrichment of CD4 in LDTI of resting lymphocytes. a, a total membrane fraction obtained from 1 × 109 surface biotinylated lymphocytes was homogenized in a buffer containing: 0, 0.02, 0.1, or 1% Triton X-100 as indicated and subjected to sucrose gradient density centrifugation. An aliquot of each fraction (10 μl) was resolved by SDS-PAGE and transferred to nitrocellulose. Fraction 1 corresponds to the top of the gradient. Biotinylated proteins were visualized by blotting with streptavidin-horseradish peroxidase. Particulate material from each fraction was also collected by centrifugation, resuspend in 30 μl, and analyzed by SDS-PAGE/Western blotting using a monoclonal antibody direct against CD4. Inset, 3 μg of fraction 5 obtained from 1% Triton gradient was immunoprecipitated with 0.5 μg of anti-CD4 IgG and subjected to SDS-PAGE and streptavidin-horseradish peroxidase blotting. For comparison, 10 μl from fraction 5 of the same gradient was analyzed as in parallel. b, 1 × 109 lymphocytes were used to prepare a particulate total membrane fraction, homogenized in a buffer containing 1% Triton X-100, and subjected to sucrose density gradient centrifugation. Twelve fractions of equal volume were collected, and their protein content was determined. The protein profile is expressed as a percentage of the total particulate loaded (2 mg). The distribution of CD4 was obtained by densitometric analysis of CD4 immunoblots. c andd, CD4 analysis in cell fractionation representing sequential steps of CD4 enrichment, starting from total cell lysate.T, total cell lysate; M, total membrane fraction (or particulate). c, values were derived from densitometry of band intensity relative to the total cell lysate (total lysate value = 1). CD4 fold enrichment was determined by the amounts used for normalized CD4 expression/relative fraction protein content (which is expressed as a percentage of the total protein). M andLDTI represent 15 and 0.2% of total protein, respectively.d, same CD4 band intensity was obtained with 50 μg of total lysate, 5 μg of total membranes, and 1 μg of the LDTI microdomains. A representative blot is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Treatment of the membrane fraction with increasing concentrations of Triton X-100 resulted in differential solubilization of the plasma membrane. As expected, in the absence of detergent, all of the biotin-labeled cell surface proteins partitioned exclusively at the bottom of the g" @default.
• W1995030412 created "2016-06-24" @default.
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• W1995030412 date "1999-05-01" @default.
• W1995030412 modified "2023-09-26" @default.
• W1995030412 title "Phorbol Ester-induced Disruption of the CD4-Lck Complex Occurs within a Detergent-resistant Microdomain of the Plasma Membrane" @default.
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