- Email: [email protected]
Interleukin-2α Receptor in Membrane Lipid Rafts
Interleukin-2␣ Receptor in Membrane Lipid Rafts Q.-R. Li, J. Ma, H. Wang, and J.-S. Li ABSTRACT Acute allograft rejection is driven by production of cytokines such as interleukin(IL)-2 that activate and expand alloreactive T cells by ligating high-affinity IL-2␣ receptors. Lipid rafts on the membranes of T cells may be the functional microdomains for IL-2␣ receptor. Flow cytometric analysis was performed for expression of CD25 (IL-2␣ receptor) on the surface of T cells. Lipid rafts were isolated by discontinuous sucrose density gradient ultracentrifugation. The localization of IL-2R␣ in fractions isolated from sucrose density gradients was determined by immunoblotting and detected by chemiluminescence. Cells were stimulated with IL-2 and expression of CD25 (IL-2␣ receptor) on the surface of T cells was 37.08%. Immunoblot analysis of fractions from sucrose gradients revealed that a large proportion of IL-2R␣ was localized in lipid rafts. Lipid rafts are the functional microdomains for IL-2␣ receptors.
I
NTERLEUKIN-2 (IL-2) exerts multiple biological functions, including serving as a T-cell growth factor, and it is produced and secreted primarily by activated T cells driving the expansion of antigen-specific cells. Acute renal allograft rejection is mediated by T cells. T cells expressing cell surface IL-2 receptors (IL-2R) were found in renal allograft.1 T cells in the graft express an increased number of IL-2R. The monitoring of plasma-soluble IL-2R concentrations has been proposed in organ transplantation, especially to detect early manifestations of rejection.2 In organ transplantation, immune activation occurs in various circumstances such as rejections and infections. Up-regulated intragraft gene expression of IL-2R molecules was observed during acute rejection episodes.3 IL-2 receptors are composed of three subunit chains: IL-R␣, IL-2R, and IL-2R␥c. IL-2R␣ (also known as CD25) is a 55-kDa transmembrane glycoprotein with 251 amino acids, which expresses only on activated T cells, and has become an important therapeutic target. IL-2R expression is very important for acute allograft. Increased levels of IL-2R␣ in biological fluids correlate with increased T- and B-cell activation and immune system activation. A correlation has been suggested between levels of IL-2 sR␣ in serum with the onset of rejection episodes in allograft recipients and a correlation of levels of IL-2R␣ in serum with disease activity in autoimmune and infectious disorders as well as in transplantation rejection.4 – 6 The IL-2R signaling pathway plays the central role in mediation of the immune response, so the monitoring of increased levels of IL-2 and IL-2R occurring in the portion of the IL-2␣ in serum shows
promise as a predictor for the onset of rejection in recipients. Lipid rafts are liquid-ordered functional membrane microdomains, which are characterized by a high content of proteins, cholesterol, and sphingolipids. Such lipids spontaneously aggregate to form liquid-ordered membrane regions, and they are insoluble in non-ionic detergents as detergent-resistant membrane domains. Simons and Toomre confirmed that lipid rafts played a role in TCR/ CD3-induced signaling.7 Biochemical analysis of the protein composition of purified lipid rafts in a large number of different cell types shows a striking concentration of signaling molecules within lipid rafts.8 This compartmentalization may have functional implications. Lipid rafts may also be involved in regulating signaling through multiple cytokine receptors.9 It is still not very clear about cytokine receptor signaling in lipid rafts. Similar to other receptors, IL-2R is not randomly distributed in the lipid bilayer, and they appear to form preexisting complexes on the surfaces of T cells.10 Lipid rafts maybe be platforms of plasma membrane From the Research Institute of General Surgery, Nanjing General Hospital of Nanjing Command, Nanjing, People’s Republic of China. Supported by National “973” Key Basic Research Project (2003CB515502) and Science Grant from Nanjing General Hospital (2004034). Address reprint requests to Qiurong Li, Research Institute of General Surgery, Nanjing General Hospital of Nanjing Command, Nanjing 210002, People’s Republic of China. E-mail: [email protected]
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.03.115
Transplantation Proceedings, 37, 2395–2397 (2005)
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clustering of IL-2R chains. In the present study, we used flow cytometry to determine CD25 expression on the surface of T cells. We further investigated whether IL-2R␣ chains localize in lipid rafts and the relationship of IL-2R␣ chains to lipid rafts in T cells in order to characterize the involvement of lipid rafts in IL-2R␣. MATERIALS AND METHODS Materials and Antibodies All chemicals were from Sigma unless stated otherwise. RPMI 1640 medium and bovine calf serum were from Invitrogen Co (Grand Island, NY, USA). Protease inhibitor cocktail tablets and BM chemiluminescence Western blotting kit were obtained from Roche Diagnostic Co (Indianapolis, Ind,: USA). The goat antihuman IL-2R␣ antibody from R&D System Inc (Minneapolis, Minn, USA) was used for Western blotting. The rabbit anti-goat IgG (H&L) horseradish peroxidase conjugated antibody was obtained from Chemicon International Inc (Temecula, Calif, USA). FITC-conjugated mouse anti-human CD25 (IL-2R␣) mAbs and isotype-matched nonreactive mouse IgG1 antibody were from Becton Dickinson Biosciences (San Diego, Calif, USA).
Cell Culture The human T-cell line Jurkat E6-1 (American Type Culture Collection, Manassas, Va, USA) was grown under standard conditions in RPMI 1640 medium supplemented with 10% heatinactivated bovine calf serum and penicillin/streptomycin (100 U/mL and 100 g/mL, respectively; Sigma) at 37°C with 5% CO2. After stimulation with 400 U/mL IL2 for 30 minutes cells were pelleted and lysed.
Isolation of Lipid Rafts Lipid rafts were isolated from Jurkat T cells as previously described11 by discontinuous sucrose density gradient ultracentrifugation. Cells were washed three times in Hanks’ balanced salt solution and lysed at 2.5 ⫻ 107 cells/mL in TKM buffer (50 mmol/L Tris, pH 7.4, 25 mmol/L KCl, 5 mmol/L MgCl2, and 1 mmol/L ethylenediamine-tetraacetic acid) containing 1% Brij58 and protease inhibitors cocktail tablets solution (0.12 mg antipain-HCl, 20 g bestatin, 40 g chymostatin, 0.12 mg E-64, 20 g leupeptin, 20 g pepstatin, 0.12 mg phosphoramidon, 0.8 mg pefabloc, 40 g aprotinin). Lysates were incubated on ice for 30 minutes, mixed
Fig 1.
with an equal volume of 80% sucrose in TKM, and overlaid with 5.5 mL 36% sucrose followed by 2.5 mL 5% sucrose. The gradients were subjected to ultracentrifugation at 250 000g at 4°C for 18 hours in a Optima L-80XP ultracentrifuge (Beckman Coulter Inc); 1 mL of fractions was collected from the top of the gradients.
Flow Cytometry Flow cytometric analysis was performed for CD25 expression on the surface of T cells. The cells were washed twice with 0.01 mol/L phosphate-buffered saline (PBS) pH 7.4 buffer and the cells were suspended with 50 L PBS buffer and incubated in dark at room temperature for 30 minutes with FITC-labeled mouse anti-human anti-CD25 (IL-2R␣) mAbs (Becton Dickinson Biosciences). The cells of negative control were incubated with isotype-matched nonreactive IgG1 antibody. Finally, the cells were washed twice in PBS and analyzed by flow cytometry using FACScalibur. Data was then analyzed with the CellQuest software.
Immunoblotting Protein of fractions isolated from sucrose density gradients was determined by immunoblotting. The fractions from sucrose gradients were separated electrophoretically on a Bio-Rad minigel apparatus (Bio-Rad) and were transferred to apolyvinylidene fluoride membranes (Bio-Rad). Western blot analysis was performed using polyclonal goat anti-human IL-2R␣ antibody (R&D) and rabbit anti-goat IgG (H&L) horseradish peroxidase-conjugated antibody. The membranes were developed with chemiluminescence Western blotting kit and were exposed to a Kodak X-ray film (Kodak BioMAX XAR; Eastman Kodak Co).
RESULTS
The IL-2R in T cells is mostly mediated by induction of IL-2R␣ chain (CD25) during activation. In order to determine IL-2R␣ expression in membrane lipid rafts of T cells, we initially detected IL-2R␣ (CD25) expression on the surface of T cells by FACS analysis (Fig 1). After IL-2 stimulation, the positive IL-2R␣ (CD25) expression cells was 37.08% (Fig 1b). In the present study we determined the localization of IL-2R␣ chains in plasma membrane of T cells. The cells were treated for 30 minutes with 400 U/mL IL-2 before lysis, and membranes were fractionated by sucrose density
FACS analysis of Jurkat T cells. (a) Negative control. (b) Surface expression of CD25 on Jurkat T cells.
INTERLEUKIN-2␣ RECEPTOR
gradient ultracentrifugation. Immunoblot analysis of fractions isolated from these gradients showed that IL-2R␣ was largely localized in lipid rafts fractions (Fig 2). It was not detected in soluble membranes fractions. DISCUSSION
Lipid rafts may play a role in the regulation of IL-2R signaling. As a first step in evaluating the role of lipid rafts in IL-2R signaling, we first confirmed the high level of IL-2R␣ (CD25) expression on the surface of T cells by flow cytometry (Fig 1) and determined the localization of IL2R␣ chains in membrances fractions isolated by ultracentrifugation (Fig 2). A substantial fraction of IL-2R␣ were associated with membrane lipid rafts in T cells and not found in the fractions of soluble membranes. IL-2R␣ was enriched in lipid rafts, which was consistent with the data of Matkó et al.12 Our results provide some evidence for a functional role of lipid rafts in the regulation of IL-2Rmediated signaling. Lipid rafts may also function to segregate signaling molecules. Compartmentation of the IL-2R chains in lipid rafts probably forms an active signaling complex and regulates IL-2R signaling in lipid rafts. Lipid rafts may have a regulatory function to control intermolecular interaction between IL-2R signaling components. It has been reported that several membrane receptors are inducibly recruited to or stabilized within these domains including T-cell receptor and the subsequent activation of signaling molecules enriched in rafts may facilitate signaling.13 Lipid rafts have been implicated in signaling via the T-cell receptor. Few studies were found about IL-2R signaling in lipid rafts. In the present study, immunoblot analysis of fractions from sucrose gradients demonstrated
Fig 2. Jurkat T cells were stimulated for 30 minutes with IL-2 before lysis. Lysates were subjected to discontinuous sucrose density gradient ultracentrifugation, and fractions (Fr) were collected and separated by SDS-PAGE. Localization of IL-2␣ was analyzed by immunoblotting.
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that IL-2R␣ was localized in lipid rafts. Whether 2R and IL-2R␥ molecules localize in lipid rafts or in soluble membranes and the downstream signal molecules such as tyrosine phosphorylation of JAK1 and JAK3 occurred in lipid rafts or in soluble membranes fractions require investigation. Our study is critical to understand the molecular events associated with their beneficial clinical effect and provides data for the onset of rejection. REFERENCES 1. Touraine F, Malcus C, Pouteil-Noble C, et al: Soluble interleukin-2 receptor (SIL-2R) in renal and pancreatic transplantation. Eur Cytokine Netw 2:47, 1991 2. Senitzer D, Greenstein SM, Louis P, et al: Monitoring serum IL-2R levels in cadaver renal transplantation: a prospective, blinded study. Transplant Proc 23:1279, 1991 3. Li YX, Li N, Li Y, et al: Up-regulated intragraft gene expression, ICAM-1 and IL-2R molecules, and apoptotic epithelial cells during rejection of rat small intestine allografts. Chin Med J 114:1089, 2001 4. Rubin LA, Nelson DL: The soluble interleukin-2 receptor: biology, function, and clinical application. Ann Intern Med 113: 619, 1990 5. Chrobak L: Clinical significance of soluble interleukin-2 receptor. Acta Medica 39:3, 1996 6. Zerler B: The soluble interleukin-2 receptor as a marker for human neoplasia and immune status. Cancer Cells 3:471, 1991 7. Simons K, Toomre D: Lipid rafts and signal transduction. Nat Rev 1:31, 2001 8. Wu C, Butz S, Ying Y, et al: Tyrosine kinase receptors concentrated in caveolae-like domains from neuronal plasma membrane. J Biol Chem 272:3554, 1997 9. Laura DZ, Stephen MR: Lipid rafts and little caves, compartmentalized signaling in membrane microdomains. J Biol Chem 277:28418, 2002 10. Damjanovich S, Bene L, Matko J, et al: Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: a fluorescence resonance energy transfer study. Proc Natl Acad U S A 94:13134, 1997 11. Stulnig TM, Huber J, Leitinger N, et al: Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition. J Bio Chem 277:28418, 2002 12. Matkó J, Bodnár A, Vereb G, et al: GPI-microdomains (membrane rafts) and signaling of the multi-chain interleukin-2 receptor in human lymphomal/leukemia T cell lines. Eur J Biochem 269:1199, 2002 13. Horejsi V, Drbal K, Cebecauer M, et al: GPI-microdomains: a role in signalling via immunoreceptors. Immunol Today 20:356, 1999
I
NTERLEUKIN-2 (IL-2) exerts multiple biological functions, including serving as a T-cell growth factor, and it is produced and secreted primarily by activated T cells driving the expansion of antigen-specific cells. Acute renal allograft rejection is mediated by T cells. T cells expressing cell surface IL-2 receptors (IL-2R) were found in renal allograft.1 T cells in the graft express an increased number of IL-2R. The monitoring of plasma-soluble IL-2R concentrations has been proposed in organ transplantation, especially to detect early manifestations of rejection.2 In organ transplantation, immune activation occurs in various circumstances such as rejections and infections. Up-regulated intragraft gene expression of IL-2R molecules was observed during acute rejection episodes.3 IL-2 receptors are composed of three subunit chains: IL-R␣, IL-2R, and IL-2R␥c. IL-2R␣ (also known as CD25) is a 55-kDa transmembrane glycoprotein with 251 amino acids, which expresses only on activated T cells, and has become an important therapeutic target. IL-2R expression is very important for acute allograft. Increased levels of IL-2R␣ in biological fluids correlate with increased T- and B-cell activation and immune system activation. A correlation has been suggested between levels of IL-2 sR␣ in serum with the onset of rejection episodes in allograft recipients and a correlation of levels of IL-2R␣ in serum with disease activity in autoimmune and infectious disorders as well as in transplantation rejection.4 – 6 The IL-2R signaling pathway plays the central role in mediation of the immune response, so the monitoring of increased levels of IL-2 and IL-2R occurring in the portion of the IL-2␣ in serum shows
promise as a predictor for the onset of rejection in recipients. Lipid rafts are liquid-ordered functional membrane microdomains, which are characterized by a high content of proteins, cholesterol, and sphingolipids. Such lipids spontaneously aggregate to form liquid-ordered membrane regions, and they are insoluble in non-ionic detergents as detergent-resistant membrane domains. Simons and Toomre confirmed that lipid rafts played a role in TCR/ CD3-induced signaling.7 Biochemical analysis of the protein composition of purified lipid rafts in a large number of different cell types shows a striking concentration of signaling molecules within lipid rafts.8 This compartmentalization may have functional implications. Lipid rafts may also be involved in regulating signaling through multiple cytokine receptors.9 It is still not very clear about cytokine receptor signaling in lipid rafts. Similar to other receptors, IL-2R is not randomly distributed in the lipid bilayer, and they appear to form preexisting complexes on the surfaces of T cells.10 Lipid rafts maybe be platforms of plasma membrane From the Research Institute of General Surgery, Nanjing General Hospital of Nanjing Command, Nanjing, People’s Republic of China. Supported by National “973” Key Basic Research Project (2003CB515502) and Science Grant from Nanjing General Hospital (2004034). Address reprint requests to Qiurong Li, Research Institute of General Surgery, Nanjing General Hospital of Nanjing Command, Nanjing 210002, People’s Republic of China. E-mail: [email protected]
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.03.115
Transplantation Proceedings, 37, 2395–2397 (2005)
2395
2396
LI, MA, WANG ET AL
clustering of IL-2R chains. In the present study, we used flow cytometry to determine CD25 expression on the surface of T cells. We further investigated whether IL-2R␣ chains localize in lipid rafts and the relationship of IL-2R␣ chains to lipid rafts in T cells in order to characterize the involvement of lipid rafts in IL-2R␣. MATERIALS AND METHODS Materials and Antibodies All chemicals were from Sigma unless stated otherwise. RPMI 1640 medium and bovine calf serum were from Invitrogen Co (Grand Island, NY, USA). Protease inhibitor cocktail tablets and BM chemiluminescence Western blotting kit were obtained from Roche Diagnostic Co (Indianapolis, Ind,: USA). The goat antihuman IL-2R␣ antibody from R&D System Inc (Minneapolis, Minn, USA) was used for Western blotting. The rabbit anti-goat IgG (H&L) horseradish peroxidase conjugated antibody was obtained from Chemicon International Inc (Temecula, Calif, USA). FITC-conjugated mouse anti-human CD25 (IL-2R␣) mAbs and isotype-matched nonreactive mouse IgG1 antibody were from Becton Dickinson Biosciences (San Diego, Calif, USA).
Cell Culture The human T-cell line Jurkat E6-1 (American Type Culture Collection, Manassas, Va, USA) was grown under standard conditions in RPMI 1640 medium supplemented with 10% heatinactivated bovine calf serum and penicillin/streptomycin (100 U/mL and 100 g/mL, respectively; Sigma) at 37°C with 5% CO2. After stimulation with 400 U/mL IL2 for 30 minutes cells were pelleted and lysed.
Isolation of Lipid Rafts Lipid rafts were isolated from Jurkat T cells as previously described11 by discontinuous sucrose density gradient ultracentrifugation. Cells were washed three times in Hanks’ balanced salt solution and lysed at 2.5 ⫻ 107 cells/mL in TKM buffer (50 mmol/L Tris, pH 7.4, 25 mmol/L KCl, 5 mmol/L MgCl2, and 1 mmol/L ethylenediamine-tetraacetic acid) containing 1% Brij58 and protease inhibitors cocktail tablets solution (0.12 mg antipain-HCl, 20 g bestatin, 40 g chymostatin, 0.12 mg E-64, 20 g leupeptin, 20 g pepstatin, 0.12 mg phosphoramidon, 0.8 mg pefabloc, 40 g aprotinin). Lysates were incubated on ice for 30 minutes, mixed
Fig 1.
with an equal volume of 80% sucrose in TKM, and overlaid with 5.5 mL 36% sucrose followed by 2.5 mL 5% sucrose. The gradients were subjected to ultracentrifugation at 250 000g at 4°C for 18 hours in a Optima L-80XP ultracentrifuge (Beckman Coulter Inc); 1 mL of fractions was collected from the top of the gradients.
Flow Cytometry Flow cytometric analysis was performed for CD25 expression on the surface of T cells. The cells were washed twice with 0.01 mol/L phosphate-buffered saline (PBS) pH 7.4 buffer and the cells were suspended with 50 L PBS buffer and incubated in dark at room temperature for 30 minutes with FITC-labeled mouse anti-human anti-CD25 (IL-2R␣) mAbs (Becton Dickinson Biosciences). The cells of negative control were incubated with isotype-matched nonreactive IgG1 antibody. Finally, the cells were washed twice in PBS and analyzed by flow cytometry using FACScalibur. Data was then analyzed with the CellQuest software.
Immunoblotting Protein of fractions isolated from sucrose density gradients was determined by immunoblotting. The fractions from sucrose gradients were separated electrophoretically on a Bio-Rad minigel apparatus (Bio-Rad) and were transferred to apolyvinylidene fluoride membranes (Bio-Rad). Western blot analysis was performed using polyclonal goat anti-human IL-2R␣ antibody (R&D) and rabbit anti-goat IgG (H&L) horseradish peroxidase-conjugated antibody. The membranes were developed with chemiluminescence Western blotting kit and were exposed to a Kodak X-ray film (Kodak BioMAX XAR; Eastman Kodak Co).
RESULTS
The IL-2R in T cells is mostly mediated by induction of IL-2R␣ chain (CD25) during activation. In order to determine IL-2R␣ expression in membrane lipid rafts of T cells, we initially detected IL-2R␣ (CD25) expression on the surface of T cells by FACS analysis (Fig 1). After IL-2 stimulation, the positive IL-2R␣ (CD25) expression cells was 37.08% (Fig 1b). In the present study we determined the localization of IL-2R␣ chains in plasma membrane of T cells. The cells were treated for 30 minutes with 400 U/mL IL-2 before lysis, and membranes were fractionated by sucrose density
FACS analysis of Jurkat T cells. (a) Negative control. (b) Surface expression of CD25 on Jurkat T cells.
INTERLEUKIN-2␣ RECEPTOR
gradient ultracentrifugation. Immunoblot analysis of fractions isolated from these gradients showed that IL-2R␣ was largely localized in lipid rafts fractions (Fig 2). It was not detected in soluble membranes fractions. DISCUSSION
Lipid rafts may play a role in the regulation of IL-2R signaling. As a first step in evaluating the role of lipid rafts in IL-2R signaling, we first confirmed the high level of IL-2R␣ (CD25) expression on the surface of T cells by flow cytometry (Fig 1) and determined the localization of IL2R␣ chains in membrances fractions isolated by ultracentrifugation (Fig 2). A substantial fraction of IL-2R␣ were associated with membrane lipid rafts in T cells and not found in the fractions of soluble membranes. IL-2R␣ was enriched in lipid rafts, which was consistent with the data of Matkó et al.12 Our results provide some evidence for a functional role of lipid rafts in the regulation of IL-2Rmediated signaling. Lipid rafts may also function to segregate signaling molecules. Compartmentation of the IL-2R chains in lipid rafts probably forms an active signaling complex and regulates IL-2R signaling in lipid rafts. Lipid rafts may have a regulatory function to control intermolecular interaction between IL-2R signaling components. It has been reported that several membrane receptors are inducibly recruited to or stabilized within these domains including T-cell receptor and the subsequent activation of signaling molecules enriched in rafts may facilitate signaling.13 Lipid rafts have been implicated in signaling via the T-cell receptor. Few studies were found about IL-2R signaling in lipid rafts. In the present study, immunoblot analysis of fractions from sucrose gradients demonstrated
Fig 2. Jurkat T cells were stimulated for 30 minutes with IL-2 before lysis. Lysates were subjected to discontinuous sucrose density gradient ultracentrifugation, and fractions (Fr) were collected and separated by SDS-PAGE. Localization of IL-2␣ was analyzed by immunoblotting.
2397
that IL-2R␣ was localized in lipid rafts. Whether 2R and IL-2R␥ molecules localize in lipid rafts or in soluble membranes and the downstream signal molecules such as tyrosine phosphorylation of JAK1 and JAK3 occurred in lipid rafts or in soluble membranes fractions require investigation. Our study is critical to understand the molecular events associated with their beneficial clinical effect and provides data for the onset of rejection. REFERENCES 1. Touraine F, Malcus C, Pouteil-Noble C, et al: Soluble interleukin-2 receptor (SIL-2R) in renal and pancreatic transplantation. Eur Cytokine Netw 2:47, 1991 2. Senitzer D, Greenstein SM, Louis P, et al: Monitoring serum IL-2R levels in cadaver renal transplantation: a prospective, blinded study. Transplant Proc 23:1279, 1991 3. Li YX, Li N, Li Y, et al: Up-regulated intragraft gene expression, ICAM-1 and IL-2R molecules, and apoptotic epithelial cells during rejection of rat small intestine allografts. Chin Med J 114:1089, 2001 4. Rubin LA, Nelson DL: The soluble interleukin-2 receptor: biology, function, and clinical application. Ann Intern Med 113: 619, 1990 5. Chrobak L: Clinical significance of soluble interleukin-2 receptor. Acta Medica 39:3, 1996 6. Zerler B: The soluble interleukin-2 receptor as a marker for human neoplasia and immune status. Cancer Cells 3:471, 1991 7. Simons K, Toomre D: Lipid rafts and signal transduction. Nat Rev 1:31, 2001 8. Wu C, Butz S, Ying Y, et al: Tyrosine kinase receptors concentrated in caveolae-like domains from neuronal plasma membrane. J Biol Chem 272:3554, 1997 9. Laura DZ, Stephen MR: Lipid rafts and little caves, compartmentalized signaling in membrane microdomains. J Biol Chem 277:28418, 2002 10. Damjanovich S, Bene L, Matko J, et al: Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: a fluorescence resonance energy transfer study. Proc Natl Acad U S A 94:13134, 1997 11. Stulnig TM, Huber J, Leitinger N, et al: Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition. J Bio Chem 277:28418, 2002 12. Matkó J, Bodnár A, Vereb G, et al: GPI-microdomains (membrane rafts) and signaling of the multi-chain interleukin-2 receptor in human lymphomal/leukemia T cell lines. Eur J Biochem 269:1199, 2002 13. Horejsi V, Drbal K, Cebecauer M, et al: GPI-microdomains: a role in signalling via immunoreceptors. Immunol Today 20:356, 1999