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First published online 11 December 2002
doi: 10.1242/jcs.00254

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Phosphorylation of FcRIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts

¹ Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland
² Universität Bielefeld, Fakultät für Chemie, Biochemie II, 33615 Bielefeld, Germany

View larger version (63K): [in a new window]   Fig. 1. Cholesterol and Src family kinases control the assembly of FcRII caps and tyrosine phosphorylation of the receptor in U937 cells. (A-F) Localization of FcRII on the cell surface. (A) Cells exposed to IV.3 mouse anti-FcRII, fixed and postlabeled with anti-mouse FITC-IgG. (B) Cells treated at 0°C with IV.3 mouse anti-FcRII and anti-mouse FITC-IgG (crosslinking). (C) Capping of crosslinked FcRII after 10 minutes of cell warming at 20°C. (D) Cells pretreated with 4 mM CDX (1 hour, 37°C) followed by crosslinking of FcRII at 0°C and (E) cells after subsequent incubation for 10 minutes at 20°C. (F) Reconstitution of FcRII cap assembly after reincorporation of cholesterol (30 minutes, 37°C) into cells pretreated with 4 mM CDX. Bar, 10 µm. (G) Quantification of FcRII cap formation as a function of cholesterol content. Bars on the left side depict a dose-dependent inhibition of FcRII capping by CDX treatment (1 hour, 37°C). Bars on the right side show the assembly of FcRII caps in cholesterol-depleted cells after 30 minutes of cholesterol reincorporation (+) or without the reincorporation (-). (-•-) Cholesterol content. (H) Tyrosine phosphorylation (PY) of FcRII immunoprecipitated from whole lysates of U937 cells (upper panel). Cells were either untreated with antibodies or exposed at 0°C to IV.3 anti-FcRII alone (non-crosslinked) or incubated at 0°C with mouse anti-FcRII and goat anti-mouse IgG (crosslinking). Prior to FcRII crosslinking, cells were preincubated with 8 µM herbimycin A (Herb), 5 mM CDX and 1 mM HMA or without inhibitors (control, Ctrl). A subset of the control cells was warmed at 20°C for 10 and 20 minutes to induce the formation of FcRII caps (capping). 52 is a molecular mass standard in kDa. Arrowhead indicates tyrosine phosphorylated FcRII. A corresponding part of the membrane was reprobed with rabbit anti-FcRII to reveal amounts of the precipitated receptor (lower panel). (I) Influence of herbimycin A (Herb), PP1, HMA, BPA, piceatannol (PCT) and wortmannin (Wort) on FcRII cap assembly. Results are the mean±s.e.m. of three to five experiments. (J) In vivo treatment of cells with PP1 and piceatannol led to the inhibition of Lyn and Syk activity. Cells were treated with 15 µM PP1 and 25-100 µM piceatannol (PCT) before (30 minutes) and during FcRII crosslinking. Syk and Lyn kinases were immunoprecipitated from lysates of the cells and the kinase assay was performed on the obtained immunocomplexes. The samples were blotted with anti-PY to reveal autophosphorylation of Syk and Lyn (arrowheads) and later reprobed with rabbit anti-Syk and mouse anti-Lyn IgG to indicate the amounts of the precipitated kinases. Lanes `non-crosslinked' show the autophosphorylation of the kinases in cells exposed to IV.3 anti-FcRII only. The level of Lyn and Syk autophosphorylation was estimated densitometrically in relation to the kinase content and is shown over corresponding lanes (the mean from two experiments). Lane "-", control of immunoprecipitation - cell lysates supplemented with Pansorbin only.

View larger version (51K): [in a new window]   Fig. 2. Distribution of Lyn, CD55, TfR and FcRII in density gradient fractions. U937 cells unexposed to inhibitors (controls, Ctrl) or preincubated with 1 mM HMA, 0.3 mM BPA or 5 mM CDX were lysed and fractionated on a density gradient in the presence of 0.2% TX-100. Fractions were immunoblotted for the presence of Lyn, CD55 and TfR (A) or biotin-labeled IV.3 anti-FcRII reflecting the receptor distribution (B). In B, the distribution of cross-linked (+) and non-crosslinked (-) FcRII is shown; in A, proteins of fractions derived prior to FcRII labeling are demonstrated. Plots display the results of densitomeric analysis of the blots shown above. The estimated amounts of an individual protein in fractions are expressed as a percentage of this total protein content in the whole gradient. Symbols used for visualization of various experimental conditions in plots of (A) and (B) are shown at the bottom of panel (B). The results are representative of three to five separate experiments.

View larger version (116K): [in a new window]   Fig. 3. Patches and caps of FcRII colocalize with CD55 but not with TfR. Distribution of FcRII (A, red) and CD55 (B, green) during FcRII crosslinking. (C) The merged image of A and B shows colocalization of CD55 and FcRII in receptor patches (yellow). (D) The merged image of FcRII (red) and CD55 (green) reveals the colocalization of the proteins in FcRII cap (yellow). Distribution of FcRII (F, red) and TfR (G, green) during FcRII crosslinking. (H) Merged images of FcRII (red) and TfR (green) show the separation of the proteins during FcRII patching. E shows representative line profiles of intensity of fluorescent labeling of crosslinked FcRII and CD55 (upper plot) and crosslinked FcRII and TfR (lower plot). Seven such profiles were generated for each analyzed cell to estimate the protein colocalization. Bar, 5 µm.

View larger version (24K): [in a new window]   Fig. 4. Crosslinked FcRII in U937 cells associates with Lyn and undergoes tyrosine phosphorylation in DRMs. (A) Tyrosine phosphorylation (PY) of proteins in OptiPrep gradient fractions. Cells were exposed to 1 mM HMA, 100 µM piceatannol, 15 µM PP1 or 5 mM CDX or left untreated (control) before crosslinking of FcRII with biotin-labeled IV.3 mouse anti-FcRII and goat anti-mouse IgG. For the analysis, 12 µl of gradient fractions 1-2 and only 4 µl of fraction 6 were used for a clearer visualization of proteins. Molecular mass standards are shown in kDa. An arrowhead marks the 40 kDa phosphoprotein corresponding to FcRII. Dots indicate proteins whose phosphorylation was significantly reduced by piceatannol. (B) Immunoprecipitation of FcRII from the gradient fractions (200 µl each). Immunoprecipitates were immunoblotted to simultaneously detect biotin-labeled anti-FcRII, reflecting the presence of the receptor (upper panel) and tyrosine-phosphorylated proteins (PY) (middle panel). An arrowhead points to FcRII; small arrows point to a doublet of 53/56 kDa phosphoproteins. The blots from the middle panel were reprobed with rabbit anti-Lyn (lower panel). The data represent one out of three experiments.

View larger version (68K): [in a new window]   Fig. 5. Tyrosine phosphorylation of FcRIIA expressed in BHK cells is required for translocation of the crosslinked receptor. (A) Comparison of phosphorylation of wild-type (wt) FcRIIA and mutant Y298F FcRIIA in cells after receptor crosslinking at 0°C and after subsequent warming of the cells for 10-20 minutes at 20°C (capping). Distribution of biotin-labeled IV.3 anti-FcRII reflects the amounts of the receptor in the blot, whereas actin labeling demonstrates equal loading of the proteins. On the left, molecular mass standards are shown in kDa. (B) Tyrosine phosphorylation of wild-type FcRIIA, induced by receptor crosslinking (Ctrl), is inhibited by 10 µM PP1 and 1 mM HMA. The blot was reprobed with anti-actin to demonstrate that equal amounts of proteins were loaded on the gel. Arrowheads indicate tyrosine-phosphorylated FcRIIA in A and B. (C) Formation of cap-like structures by crosslinked wild-type FcRIIA and Y298F FcRIIA in BHK transfectants. The results are the means±s.e.m. from four experiments. (D) Colocalization of crosslinked wild-type FcRIIA with tyrosine-phosphorylated proteins and actin filaments during receptor crosslinking (0°C) and formation of cap-like structures (10 minutes at 20°C). Inserts in the `crosslinking' row show a magnified fragment of the cell, which reveals significant colocalization of crosslinked FcRIIA and phosphotyrosine-bearing proteins. (E) Crosslinking of mutant Y298F FcRIIA at 0°C did not induce tyrosine phosphorylation of proteins and no cap-like structures are formed after 10 minutes at 20°C. In D and E, the top panels show cells fixed directly after binding of IV.3 anti-FcRII, which reveals diffuse distribution of the non-crosslinked receptors on the cell surface and low level of phosphotyrosine-bearing proteins inside the cells. Bar, 15 µm.

View larger version (86K): [in a new window]   Fig. 6. Wild-type (wt) FcRIIA but not Y298F FcRIIA mediates spreading of BHK transfectants. (A) Cells were plated on coverslips coated with IV.3 anti-FcRII. In control samples, BSA or fibronectin (Fn) were used as a substratum. Where indicated, cells were pretreated with 15 µM cytochalasin B (CB), 10 µM PP1, 1 mM HMA or 8 mM MCDX. Cells after 2 hours (or 5 hours, as indicated) of spreading are shown. Organization of actin filaments visualized with phalloidin-TRITC is demonstrated in the lower panel. Bars, 20 µm. (B) Quantification of spreading of BHK cells transfected with wild-type FcRIIA and Y298F FcRIIA on IV.3-coated substratum 2 hours after plating. Spreading of cells transfected with wild-type receptor was inhibited after pretreatment with 15 µM cytochalasin B, 10 µM PP1, 1 mM HMA or 8 mM MCDX. The results are the means±s.e.m. from two experiments. (C) Tyrosine-phosphorylated FcRIIA is marked by an arrowhead. Phosphorylation of wild-type FcRIIA was induced by spreading of BHK cells on IV.3 but not on fibronectin-coated substratum. No phosphorylation of Y298F FcRIIA was detected during spreading of the receptor-expressing cells on IV.3-coated substratum. Cell spreading was carried out for 0.5, 2 and 3 hours. The blot was reprobed with anti-actin to reveal equal loading of proteins. Molecular mass standards are shown on the left in kDa.