Tyrosine phosphorylation of Fc ⑀ RI before and after cholesterol depletion/repletion. IgE-sensitized cells were sequen- tially treated with ( ϩ ) or without (–) 10 mM M ␤ CD for 1 h at 37 Њ C, then for 2 h at 37 Њ C with 3 mM M ␤ CD (*) (lanes 5 and 6), with 300 mM M ␤ CD/cholesterol (8:1, mol/mol) diluted as indicated (†) (lanes 7–12), or with buffer alone (lanes 1–4). Washed cells were stimulated for 2 min with ( ϩ ) or without ( Ϫ ) 1 ␮ g/ml DNP-BSA at 37 Њ C, then lysed and analyzed by antiphosphotyrosine Western blot analysis as described for Fig. 1 a. 

Contexts in source publication

Context 1

... indicating that lateral mobility is not im- peded by cholesterol depletion; however, Lyn does not re- distribute with IgE–Fc ⑀ RI under these conditions (Fig. 4 d). As indicated in the first line of Table I, these differences are statistically significant when quantified by cross correlation analysis of multiple cells. Thus, cross-link– dependent interactions between Fc ⑀ RI and Lyn on the cell surface are largely prevented by cholesterol depletion, consistent with the loss of interactions of these proteins with DRMs in the sucrose gradient analyses of lysed cells described above. Fig. 4 e shows that, as previously observed (Pierini et al., 1996), cross-linking of IgE–Fc ⑀ RI at the cell surface results in co-redistribution of the GD 1b ganglioside that is labeled by Cy3-AA4 mAb. For M ␤ CD-treated cells, we find that co-redistribution of this outer leaflet DRM marker with cross-linked IgE–Fc ⑀ RI is reduced compared with control cells but not completely disrupted as was the case for Lyn. Fig. 4 f shows an example of this variability, in which one cell exhibits partial co-redistribution of the labeled gangli- oside, and the other shows a complete lack of co-redistribution with patched IgE–Fc ⑀ RI. Under these conditions, Յ 20% of the cells exhibited detectable co-redistribution of labeled ganglioside, whereas no detectable co-redistribution of Lyn with IgE–Fc ⑀ RI patches was observed. From these results, it appears that the interaction between Lyn and Fc ⑀ RI is more sensitive to cholesterol depletion than is the interaction between the ganglioside and Fc ⑀ RI in the intact cells, although both are substantially prevented. The difference observed may be related to the greater sensitivity of the Lyn–DRM interactions than GD 1b –DRM interactions, as shown in Fig. 3 (see Discussion). As a further control, we compared the distribution of the transferrin receptor (CD71) to FITC-IgE–Fc ⑀ RI cross- linked under the same conditions as above. Previous stud- ies showed that this transmembrane protein does not associate with isolated DRMs (Melkonian et al., 1999), nor does it co-redistribute with other DRM-associated proteins when simultaneously but separately cross-linked on BHK and Jurkat T cells (Harder et al., 1998). As seen in Fig. 4, g and h, TfRs do not co-redistribute with cross- linked IgE–Fc ⑀ RI, and they remain evenly distributed around the periphery of the cell, often in tiny clusters that may reflect interactions with coated pits. Quantitative analysis of the cross correlation of TfR with cross-linked IgE–Fc ⑀ RI show no appreciable colocalization between these molecules whether or not the cells have been depleted of cholesterol (see Table I). These results support the significance of the co-redistribution of Lyn with cross- linked IgE–Fc ⑀ RI described above, as well as its inhibition by cholesterol depletion with M ␤ CD. To investigate the reversibility of cholesterol effects on the functional and structural interactions of Fc ⑀ RI with Lyn, we restored cholesterol levels in M ␤ CD-treated cells by incubating them with cholesterol–M ␤ CD complexes. The efficiency of repletion is dependent upon the incubation period of the cells with the complex, the molar ratio of cholesterol to M ␤ CD, and the final concentration of M ␤ CD (Christian et al., 1997; Sheets, E.D., unpublished results). To optimize repletion, we used several dilutions of cholesterol–M ␤ CD complexes prepared as described in Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control samples) or incubated with M ␤ CD to lower cholesterol levels as described above. During the second step, the cells were incubated for 2 h at 37 Њ C at the indicated dilution of cholesterol/M ␤ CD, 3 mM M ␤ CD only, or buffer only. We found that (a) the cholesterol levels of cells depleted by exposure to M ␤ CD in the first step did not change during the subsequent incubation in the absence of M ␤ CD; (b) the presence of 3 mM of M ␤ CD during the second step also did not cause additional cholesterol depletion, nor did it alter the distribution of IgE–Fc ⑀ RI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3–6 mM M ␤ CD; 8:1, mol/mol M ␤ CD/cholesterol), the cholesterol content of the repleted cells was 3.0–3.5-fold higher than that in the untreated control cells. Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed de- tectably during the depletion/repletion treatments (data not shown). As shown in Fig. 5, repletion of cholesterol in M ␤ CD- treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown, maximal recovery of stimulated tyrosine phosphorylation of Fc ⑀ RI ␤ and other bands was achieved when 1:50 dilution of the preformed 8:1 M ␤ CD/cholesterol complexes was used to give a final concentration of 6 mM M ␤ CD during the repletion step (Fig. 5, lane 10). As seen in Fig. 5, lane 6, cells that had been treated with M ␤ CD alone during both the depletion and repletion steps have no detectable ␤ phosphorylation, and only a very small amount of stimulated tyrosine phosphorylation is seen in the higher molecular weight bands. Similar results to these were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE– Fc ⑀ RI was determined to be 77 Ϯ 4% ( n ϭ 5), and, based upon the proportional relationship between receptor number and ␤ phosphorylation (above), this leads us to expect that optimal restoration of Fc ⑀ RI ␤ phosphorylation should be ف 23% of the stimulated control in Fig. 5, lane 2. The somewhat smaller restoration that is apparent (Fig. 5, lanes 10 and 12) suggests that other factors, such as the loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum restoration achievable (see Discussion). Repletion of cholesterol also results in restoration of cross-link–dependent association of IgE–Fc ⑀ RI with isolated DRMs. When lysates of cholesterol-repleted cells are analyzed on sucrose gradients, cross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭝ ) migrates to the low density sucrose region, whereas uncross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭡ ) is found in the 40% sucrose region, similar to the gradient distributions from control cells (Fig. 3 a, ᭺ and ᭹ ). Cross- linked IgE–Fc ⑀ RI from cholesterol-repleted cells migrate at slightly lower densities in the sucrose gradients than this complex in the control cells, suggesting that the average density of DRMs in repleted cells is lower than in control cells, possibly due to a decrease in the protein/lipid ratio resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates to the low density region of the gradient (fractions 1–6) after cholesterol repletion in cells with both cross-linked and uncross-linked Fc ⑀ RI. These results, in parallel with the restoration of stimulated Fc ⑀ RI tyrosine phosphorylation (Fig. 5), provide strong evidence that cholesterol is important for functional coupling of Fc ⑀ RI with Lyn and for their mutual association with DRMs. Our results demonstrate that cholesterol plays a critical role in the initial step of Fc ⑀ RI signaling: antigen-stimu- lated tyrosine phosphorylation of this receptor by the Src family tyrosine kinase Lyn. In parallel with loss of this stimulated phosphorylation (Fig. 1), reduction of cellular cholesterol by M ␤ CD causes the loss of association of both Lyn and cross-linked Fc ⑀ RI with DRMs isolated after cell lysis by TX-100 (Fig. 3). Restoration of the cholesterol content of the depleted cells using preformed cholesterol–M ␤ CD complexes restores the association of Lyn and cross-linked IgE–Fc ⑀ RI with DRMs (Fig. 3) and also causes partial restoration of antigen-stimulated tyrosine phosphorylation in the cells (Fig. 5). These results support the hypothesis that interactions of cross-linked IgE–Fc ⑀ RI with DRMs are important for the initial coupling of Fc ⑀ RI and Lyn that results in receptor phosphorylation. On the cell surface, the association of Lyn with aggregated IgE– Fc ⑀ RI is lost as the result of cholesterol depletion (Fig. 4), indicating that the interactions detected in isolated DRMs are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked Fc ⑀ RI with Lyn as the basis for association of receptors with DRMs, and they support the view that the L o structure of the plasma membrane is important for Fc ⑀ RI–Lyn interactions. An initially surprising finding in our studies is the appar- ently selective loss of Fc ⑀ RI and outer leaflet plasma membrane components of DRMs due to cholesterol depletion by M ␤ CD. As indicated by Western blot analysis and fluorescence microscopy, there is a smaller loss of Lyn due to cholesterol depletion, and there is no detectable loss of other cellular proteins by silver stain analysis of whole cell lysates (data not shown). The mechanism by which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles containing these components may pinch off from the cells in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by Ilangumaran and Hoessli (1998), a similar preferential release of DRM components by M ␤ CD treatment was characterized in lymphocytes and endothelial cells, and evidence for release of these components in membrane vesicles was described. Our results suggest that Fc ⑀ RI may preferentially associate with DRM components on intact cells even in the absence of receptor cross-linking. Consistent with this, Basciano et al. showed that pre-binding of AA4 mAb or its Fab fragment to the ␣ -galactosyl GD 1b antigen on RBL-2H3 cells can effectively inhibit the ...

Context 2

... of Fc ⑀ RI with Lyn, we restored cholesterol levels in M ␤ CD-treated cells by incubating them with cholesterol–M ␤ CD complexes. The efficiency of repletion is dependent upon the incubation period of the cells with the complex, the molar ratio of cholesterol to M ␤ CD, and the final concentration of M ␤ CD (Christian et al., 1997; Sheets, E.D., unpublished results). To optimize repletion, we used several dilutions of cholesterol–M ␤ CD complexes prepared as described in Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control samples) or incubated with M ␤ CD to lower cholesterol levels as described above. During the second step, the cells were incubated for 2 h at 37 Њ C at the indicated dilution of cholesterol/M ␤ CD, 3 mM M ␤ CD only, or buffer only. We found that (a) the cholesterol levels of cells depleted by exposure to M ␤ CD in the first step did not change during the subsequent incubation in the absence of M ␤ CD; (b) the presence of 3 mM of M ␤ CD during the second step also did not cause additional cholesterol depletion, nor did it alter the distribution of IgE–Fc ⑀ RI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3–6 mM M ␤ CD; 8:1, mol/mol M ␤ CD/cholesterol), the cholesterol content of the repleted cells was 3.0–3.5-fold higher than that in the untreated control cells. Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed de- tectably during the depletion/repletion treatments (data not shown). As shown in Fig. 5, repletion of cholesterol in M ␤ CD- treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown, maximal recovery of stimulated tyrosine phosphorylation of Fc ⑀ RI ␤ and other bands was achieved when 1:50 dilution of the preformed 8:1 M ␤ CD/cholesterol complexes was used to give a final concentration of 6 mM M ␤ CD during the repletion step (Fig. 5, lane 10). As seen in Fig. 5, lane 6, cells that had been treated with M ␤ CD alone during both the depletion and repletion steps have no detectable ␤ phosphorylation, and only a very small amount of stimulated tyrosine phosphorylation is seen in the higher molecular weight bands. Similar results to these were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE– Fc ⑀ RI was determined to be 77 Ϯ 4% ( n ϭ 5), and, based upon the proportional relationship between receptor number and ␤ phosphorylation (above), this leads us to expect that optimal restoration of Fc ⑀ RI ␤ phosphorylation should be ف 23% of the stimulated control in Fig. 5, lane 2. The somewhat smaller restoration that is apparent (Fig. 5, lanes 10 and 12) suggests that other factors, such as the loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum restoration achievable (see Discussion). Repletion of cholesterol also results in restoration of cross-link–dependent association of IgE–Fc ⑀ RI with isolated DRMs. When lysates of cholesterol-repleted cells are analyzed on sucrose gradients, cross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭝ ) migrates to the low density sucrose region, whereas uncross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭡ ) is found in the 40% sucrose region, similar to the gradient distributions from control cells (Fig. 3 a, ᭺ and ᭹ ). Cross- linked IgE–Fc ⑀ RI from cholesterol-repleted cells migrate at slightly lower densities in the sucrose gradients than this complex in the control cells, suggesting that the average density of DRMs in repleted cells is lower than in control cells, possibly due to a decrease in the protein/lipid ratio resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates to the low density region of the gradient (fractions 1–6) after cholesterol repletion in cells with both cross-linked and uncross-linked Fc ⑀ RI. These results, in parallel with the restoration of stimulated Fc ⑀ RI tyrosine phosphorylation (Fig. 5), provide strong evidence that cholesterol is important for functional coupling of Fc ⑀ RI with Lyn and for their mutual association with DRMs. Our results demonstrate that cholesterol plays a critical role in the initial step of Fc ⑀ RI signaling: antigen-stimu- lated tyrosine phosphorylation of this receptor by the Src family tyrosine kinase Lyn. In parallel with loss of this stimulated phosphorylation (Fig. 1), reduction of cellular cholesterol by M ␤ CD causes the loss of association of both Lyn and cross-linked Fc ⑀ RI with DRMs isolated after cell lysis by TX-100 (Fig. 3). Restoration of the cholesterol content of the depleted cells using preformed cholesterol–M ␤ CD complexes restores the association of Lyn and cross-linked IgE–Fc ⑀ RI with DRMs (Fig. 3) and also causes partial restoration of antigen-stimulated tyrosine phosphorylation in the cells (Fig. 5). These results support the hypothesis that interactions of cross-linked IgE–Fc ⑀ RI with DRMs are important for the initial coupling of Fc ⑀ RI and Lyn that results in receptor phosphorylation. On the cell surface, the association of Lyn with aggregated IgE– Fc ⑀ RI is lost as the result of cholesterol depletion (Fig. 4), indicating that the interactions detected in isolated DRMs are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked Fc ⑀ RI with Lyn as the basis for association of receptors with DRMs, and they support the view that the L o structure of the plasma membrane is important for Fc ⑀ RI–Lyn interactions. An initially surprising finding in our studies is the appar- ently selective loss of Fc ⑀ RI and outer leaflet plasma membrane components of DRMs due to cholesterol depletion by M ␤ CD. As indicated by Western blot analysis and fluorescence microscopy, there is a smaller loss of Lyn due to cholesterol depletion, and there is no detectable loss of other cellular proteins by silver stain analysis of whole cell lysates (data not shown). The mechanism by which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles containing these components may pinch off from the cells in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by Ilangumaran and Hoessli (1998), a similar preferential release of DRM components by M ␤ CD treatment was characterized in lymphocytes and endothelial cells, and evidence for release of these components in membrane vesicles was described. Our results suggest that Fc ⑀ RI may preferentially associate with DRM components on intact cells even in the absence of receptor cross-linking. Consistent with this, Basciano et al. showed that pre-binding of AA4 mAb or its Fab fragment to the ␣ -galactosyl GD 1b antigen on RBL-2H3 cells can effectively inhibit the subsequent binding of IgE to Fc ⑀ RI (Basciano et al., 1986). By varying the cell surface density of antigen-specific IgE in the range of 30–100%, we show that the loss of Fc ⑀ RI due to cholesterol depletion cannot account for the nearly complete inhibition of Fc ⑀ RI tyrosine phosphorylation that is observed in these cells. Furthermore, the partial restoration of antigen-stimulated tyrosine phosphorylation without an increase in Fc ⑀ RI expression after cholesterol repletion strengthens the evidence that cellular cholesterol critically regulates the coupling of the remaining Fc ⑀ RI and Lyn. An important observation by fluorescence microscopy is that cholesterol depletion does not prevent antigen- dependent aggregation of IgE–Fc ⑀ RI on the cell surface, even though it prevents the co-redistribution of Lyn and inhibits stimulated tyrosine phosphorylation. We previ- ously showed that the cholesterol-binding polyene antibi- otic, filipin, prevents anti-IgE–mediated patching of IgE– Fc ⑀ RI (Feder et al., 1994), indicating that it may prevent aggregation of the receptor necessary to initiate signaling. Unlike M ␤ CD, which extracts cholesterol into a water-sol- uble complex that can be washed away, filipin forms complexes with cholesterol in the membrane that appear to re- strict lateral diffusion of at least some membrane proteins, making this reagent less useful for studying the role of cholesterol in signaling by receptors that must aggregate in response to their ligands to be effective. The rapidity with which M ␤ CD can reduce cell cholesterol by substantial amounts without significantly compromising cell integrity, as evidenced by our degranulation results, and the capac- ity to restore stimulated tyrosine phosphorylation by rein- troduction of cholesterol via M ␤ CD complexes, make this an extremely valuable tool for investigating the role of cholesterol in a wide variety of receptor systems. Recent studies used M ␤ CD to investigate the role of cholesterol in signaling by other receptors. Pike and Miller (1998) showed that cholesterol depletion by M ␤ CD inhibits EGF- and bradykinin-stimulated phosphatidylinositol turnover, which can be restored by cholesterol repletion with M ␤ CD–cholesterol complexes. These receptors be- long to the families of intrinsic tyrosine kinase receptors and G protein–coupled receptors, respectively, suggesting the potentially general importance of cholesterol and the L o structure it confers on the plasma membrane in medi- ating receptor signaling. Interestingly, cholesterol depletion by M ␤ CD does not inhibit EGF-stimulated tyrosine phosphorylation of its receptor (Pike, L.J., Y. Liu, K.N. Chung, and J.A. Heuser. 1998. FASEB J . 12:A1278 [ab- str.]), which probably occurs via a transphosphorylation mechanism (Weiss and Schlessinger, 1998). Rather, cholesterol depletion appears to affect the compartmentaliza- tion of phosphatidylinositol 4,5-bisphosphate, the primary phospholipase C ...

Context 3

... is not im- peded by cholesterol depletion; however, Lyn does not re- distribute with IgE–Fc ⑀ RI under these conditions (Fig. 4 d). As indicated in the first line of Table I, these differences are statistically significant when quantified by cross correlation analysis of multiple cells. Thus, cross-link– dependent interactions between Fc ⑀ RI and Lyn on the cell surface are largely prevented by cholesterol depletion, consistent with the loss of interactions of these proteins with DRMs in the sucrose gradient analyses of lysed cells described above. Fig. 4 e shows that, as previously observed (Pierini et al., 1996), cross-linking of IgE–Fc ⑀ RI at the cell surface results in co-redistribution of the GD 1b ganglioside that is labeled by Cy3-AA4 mAb. For M ␤ CD-treated cells, we find that co-redistribution of this outer leaflet DRM marker with cross-linked IgE–Fc ⑀ RI is reduced compared with control cells but not completely disrupted as was the case for Lyn. Fig. 4 f shows an example of this variability, in which one cell exhibits partial co-redistribution of the labeled gangli- oside, and the other shows a complete lack of co-redistribution with patched IgE–Fc ⑀ RI. Under these conditions, Յ 20% of the cells exhibited detectable co-redistribution of labeled ganglioside, whereas no detectable co-redistribution of Lyn with IgE–Fc ⑀ RI patches was observed. From these results, it appears that the interaction between Lyn and Fc ⑀ RI is more sensitive to cholesterol depletion than is the interaction between the ganglioside and Fc ⑀ RI in the intact cells, although both are substantially prevented. The difference observed may be related to the greater sensitivity of the Lyn–DRM interactions than GD 1b –DRM interactions, as shown in Fig. 3 (see Discussion). As a further control, we compared the distribution of the transferrin receptor (CD71) to FITC-IgE–Fc ⑀ RI cross- linked under the same conditions as above. Previous stud- ies showed that this transmembrane protein does not associate with isolated DRMs (Melkonian et al., 1999), nor does it co-redistribute with other DRM-associated proteins when simultaneously but separately cross-linked on BHK and Jurkat T cells (Harder et al., 1998). As seen in Fig. 4, g and h, TfRs do not co-redistribute with cross- linked IgE–Fc ⑀ RI, and they remain evenly distributed around the periphery of the cell, often in tiny clusters that may reflect interactions with coated pits. Quantitative analysis of the cross correlation of TfR with cross-linked IgE–Fc ⑀ RI show no appreciable colocalization between these molecules whether or not the cells have been depleted of cholesterol (see Table I). These results support the significance of the co-redistribution of Lyn with cross- linked IgE–Fc ⑀ RI described above, as well as its inhibition by cholesterol depletion with M ␤ CD. To investigate the reversibility of cholesterol effects on the functional and structural interactions of Fc ⑀ RI with Lyn, we restored cholesterol levels in M ␤ CD-treated cells by incubating them with cholesterol–M ␤ CD complexes. The efficiency of repletion is dependent upon the incubation period of the cells with the complex, the molar ratio of cholesterol to M ␤ CD, and the final concentration of M ␤ CD (Christian et al., 1997; Sheets, E.D., unpublished results). To optimize repletion, we used several dilutions of cholesterol–M ␤ CD complexes prepared as described in Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control samples) or incubated with M ␤ CD to lower cholesterol levels as described above. During the second step, the cells were incubated for 2 h at 37 Њ C at the indicated dilution of cholesterol/M ␤ CD, 3 mM M ␤ CD only, or buffer only. We found that (a) the cholesterol levels of cells depleted by exposure to M ␤ CD in the first step did not change during the subsequent incubation in the absence of M ␤ CD; (b) the presence of 3 mM of M ␤ CD during the second step also did not cause additional cholesterol depletion, nor did it alter the distribution of IgE–Fc ⑀ RI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3–6 mM M ␤ CD; 8:1, mol/mol M ␤ CD/cholesterol), the cholesterol content of the repleted cells was 3.0–3.5-fold higher than that in the untreated control cells. Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed de- tectably during the depletion/repletion treatments (data not shown). As shown in Fig. 5, repletion of cholesterol in M ␤ CD- treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown, maximal recovery of stimulated tyrosine phosphorylation of Fc ⑀ RI ␤ and other bands was achieved when 1:50 dilution of the preformed 8:1 M ␤ CD/cholesterol complexes was used to give a final concentration of 6 mM M ␤ CD during the repletion step (Fig. 5, lane 10). As seen in Fig. 5, lane 6, cells that had been treated with M ␤ CD alone during both the depletion and repletion steps have no detectable ␤ phosphorylation, and only a very small amount of stimulated tyrosine phosphorylation is seen in the higher molecular weight bands. Similar results to these were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE– Fc ⑀ RI was determined to be 77 Ϯ 4% ( n ϭ 5), and, based upon the proportional relationship between receptor number and ␤ phosphorylation (above), this leads us to expect that optimal restoration of Fc ⑀ RI ␤ phosphorylation should be ف 23% of the stimulated control in Fig. 5, lane 2. The somewhat smaller restoration that is apparent (Fig. 5, lanes 10 and 12) suggests that other factors, such as the loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum restoration achievable (see Discussion). Repletion of cholesterol also results in restoration of cross-link–dependent association of IgE–Fc ⑀ RI with isolated DRMs. When lysates of cholesterol-repleted cells are analyzed on sucrose gradients, cross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭝ ) migrates to the low density sucrose region, whereas uncross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭡ ) is found in the 40% sucrose region, similar to the gradient distributions from control cells (Fig. 3 a, ᭺ and ᭹ ). Cross- linked IgE–Fc ⑀ RI from cholesterol-repleted cells migrate at slightly lower densities in the sucrose gradients than this complex in the control cells, suggesting that the average density of DRMs in repleted cells is lower than in control cells, possibly due to a decrease in the protein/lipid ratio resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates to the low density region of the gradient (fractions 1–6) after cholesterol repletion in cells with both cross-linked and uncross-linked Fc ⑀ RI. These results, in parallel with the restoration of stimulated Fc ⑀ RI tyrosine phosphorylation (Fig. 5), provide strong evidence that cholesterol is important for functional coupling of Fc ⑀ RI with Lyn and for their mutual association with DRMs. Our results demonstrate that cholesterol plays a critical role in the initial step of Fc ⑀ RI signaling: antigen-stimu- lated tyrosine phosphorylation of this receptor by the Src family tyrosine kinase Lyn. In parallel with loss of this stimulated phosphorylation (Fig. 1), reduction of cellular cholesterol by M ␤ CD causes the loss of association of both Lyn and cross-linked Fc ⑀ RI with DRMs isolated after cell lysis by TX-100 (Fig. 3). Restoration of the cholesterol content of the depleted cells using preformed cholesterol–M ␤ CD complexes restores the association of Lyn and cross-linked IgE–Fc ⑀ RI with DRMs (Fig. 3) and also causes partial restoration of antigen-stimulated tyrosine phosphorylation in the cells (Fig. 5). These results support the hypothesis that interactions of cross-linked IgE–Fc ⑀ RI with DRMs are important for the initial coupling of Fc ⑀ RI and Lyn that results in receptor phosphorylation. On the cell surface, the association of Lyn with aggregated IgE– Fc ⑀ RI is lost as the result of cholesterol depletion (Fig. 4), indicating that the interactions detected in isolated DRMs are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked Fc ⑀ RI with Lyn as the basis for association of receptors with DRMs, and they support the view that the L o structure of the plasma membrane is important for Fc ⑀ RI–Lyn interactions. An initially surprising finding in our studies is the appar- ently selective loss of Fc ⑀ RI and outer leaflet plasma membrane components of DRMs due to cholesterol depletion by M ␤ CD. As indicated by Western blot analysis and fluorescence microscopy, there is a smaller loss of Lyn due to cholesterol depletion, and there is no detectable loss of other cellular proteins by silver stain analysis of whole cell lysates (data not shown). The mechanism by which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles containing these components may pinch off from the cells in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by Ilangumaran and Hoessli (1998), a similar preferential release of DRM components by M ␤ CD treatment was characterized in lymphocytes and endothelial cells, and evidence for release of these components in membrane vesicles was described. Our results suggest that Fc ⑀ RI may preferentially associate with DRM components on intact cells even in the absence of receptor cross-linking. Consistent with this, Basciano et al. showed that pre-binding of AA4 mAb or its Fab fragment to the ␣ -galactosyl GD 1b antigen on RBL-2H3 cells can effectively inhibit the subsequent binding of IgE to Fc ⑀ ...

Context 4

... does it co-redistribute with other DRM-associated proteins when simultaneously but separately cross-linked on BHK and Jurkat T cells (Harder et al., 1998). As seen in Fig. 4, g and h, TfRs do not co-redistribute with cross- linked IgE–Fc ⑀ RI, and they remain evenly distributed around the periphery of the cell, often in tiny clusters that may reflect interactions with coated pits. Quantitative analysis of the cross correlation of TfR with cross-linked IgE–Fc ⑀ RI show no appreciable colocalization between these molecules whether or not the cells have been depleted of cholesterol (see Table I). These results support the significance of the co-redistribution of Lyn with cross- linked IgE–Fc ⑀ RI described above, as well as its inhibition by cholesterol depletion with M ␤ CD. To investigate the reversibility of cholesterol effects on the functional and structural interactions of Fc ⑀ RI with Lyn, we restored cholesterol levels in M ␤ CD-treated cells by incubating them with cholesterol–M ␤ CD complexes. The efficiency of repletion is dependent upon the incubation period of the cells with the complex, the molar ratio of cholesterol to M ␤ CD, and the final concentration of M ␤ CD (Christian et al., 1997; Sheets, E.D., unpublished results). To optimize repletion, we used several dilutions of cholesterol–M ␤ CD complexes prepared as described in Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control samples) or incubated with M ␤ CD to lower cholesterol levels as described above. During the second step, the cells were incubated for 2 h at 37 Њ C at the indicated dilution of cholesterol/M ␤ CD, 3 mM M ␤ CD only, or buffer only. We found that (a) the cholesterol levels of cells depleted by exposure to M ␤ CD in the first step did not change during the subsequent incubation in the absence of M ␤ CD; (b) the presence of 3 mM of M ␤ CD during the second step also did not cause additional cholesterol depletion, nor did it alter the distribution of IgE–Fc ⑀ RI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3–6 mM M ␤ CD; 8:1, mol/mol M ␤ CD/cholesterol), the cholesterol content of the repleted cells was 3.0–3.5-fold higher than that in the untreated control cells. Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed de- tectably during the depletion/repletion treatments (data not shown). As shown in Fig. 5, repletion of cholesterol in M ␤ CD- treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown, maximal recovery of stimulated tyrosine phosphorylation of Fc ⑀ RI ␤ and other bands was achieved when 1:50 dilution of the preformed 8:1 M ␤ CD/cholesterol complexes was used to give a final concentration of 6 mM M ␤ CD during the repletion step (Fig. 5, lane 10). As seen in Fig. 5, lane 6, cells that had been treated with M ␤ CD alone during both the depletion and repletion steps have no detectable ␤ phosphorylation, and only a very small amount of stimulated tyrosine phosphorylation is seen in the higher molecular weight bands. Similar results to these were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE– Fc ⑀ RI was determined to be 77 Ϯ 4% ( n ϭ 5), and, based upon the proportional relationship between receptor number and ␤ phosphorylation (above), this leads us to expect that optimal restoration of Fc ⑀ RI ␤ phosphorylation should be ف 23% of the stimulated control in Fig. 5, lane 2. The somewhat smaller restoration that is apparent (Fig. 5, lanes 10 and 12) suggests that other factors, such as the loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum restoration achievable (see Discussion). Repletion of cholesterol also results in restoration of cross-link–dependent association of IgE–Fc ⑀ RI with isolated DRMs. When lysates of cholesterol-repleted cells are analyzed on sucrose gradients, cross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭝ ) migrates to the low density sucrose region, whereas uncross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭡ ) is found in the 40% sucrose region, similar to the gradient distributions from control cells (Fig. 3 a, ᭺ and ᭹ ). Cross- linked IgE–Fc ⑀ RI from cholesterol-repleted cells migrate at slightly lower densities in the sucrose gradients than this complex in the control cells, suggesting that the average density of DRMs in repleted cells is lower than in control cells, possibly due to a decrease in the protein/lipid ratio resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates to the low density region of the gradient (fractions 1–6) after cholesterol repletion in cells with both cross-linked and uncross-linked Fc ⑀ RI. These results, in parallel with the restoration of stimulated Fc ⑀ RI tyrosine phosphorylation (Fig. 5), provide strong evidence that cholesterol is important for functional coupling of Fc ⑀ RI with Lyn and for their mutual association with DRMs. Our results demonstrate that cholesterol plays a critical role in the initial step of Fc ⑀ RI signaling: antigen-stimu- lated tyrosine phosphorylation of this receptor by the Src family tyrosine kinase Lyn. In parallel with loss of this stimulated phosphorylation (Fig. 1), reduction of cellular cholesterol by M ␤ CD causes the loss of association of both Lyn and cross-linked Fc ⑀ RI with DRMs isolated after cell lysis by TX-100 (Fig. 3). Restoration of the cholesterol content of the depleted cells using preformed cholesterol–M ␤ CD complexes restores the association of Lyn and cross-linked IgE–Fc ⑀ RI with DRMs (Fig. 3) and also causes partial restoration of antigen-stimulated tyrosine phosphorylation in the cells (Fig. 5). These results support the hypothesis that interactions of cross-linked IgE–Fc ⑀ RI with DRMs are important for the initial coupling of Fc ⑀ RI and Lyn that results in receptor phosphorylation. On the cell surface, the association of Lyn with aggregated IgE– Fc ⑀ RI is lost as the result of cholesterol depletion (Fig. 4), indicating that the interactions detected in isolated DRMs are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked Fc ⑀ RI with Lyn as the basis for association of receptors with DRMs, and they support the view that the L o structure of the plasma membrane is important for Fc ⑀ RI–Lyn interactions. An initially surprising finding in our studies is the appar- ently selective loss of Fc ⑀ RI and outer leaflet plasma membrane components of DRMs due to cholesterol depletion by M ␤ CD. As indicated by Western blot analysis and fluorescence microscopy, there is a smaller loss of Lyn due to cholesterol depletion, and there is no detectable loss of other cellular proteins by silver stain analysis of whole cell lysates (data not shown). The mechanism by which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles containing these components may pinch off from the cells in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by Ilangumaran and Hoessli (1998), a similar preferential release of DRM components by M ␤ CD treatment was characterized in lymphocytes and endothelial cells, and evidence for release of these components in membrane vesicles was described. Our results suggest that Fc ⑀ RI may preferentially associate with DRM components on intact cells even in the absence of receptor cross-linking. Consistent with this, Basciano et al. showed that pre-binding of AA4 mAb or its Fab fragment to the ␣ -galactosyl GD 1b antigen on RBL-2H3 cells can effectively inhibit the subsequent binding of IgE to Fc ⑀ RI (Basciano et al., 1986). By varying the cell surface density of antigen-specific IgE in the range of 30–100%, we show that the loss of Fc ⑀ RI due to cholesterol depletion cannot account for the nearly complete inhibition of Fc ⑀ RI tyrosine phosphorylation that is observed in these cells. Furthermore, the partial restoration of antigen-stimulated tyrosine phosphorylation without an increase in Fc ⑀ RI expression after cholesterol repletion strengthens the evidence that cellular cholesterol critically regulates the coupling of the remaining Fc ⑀ RI and Lyn. An important observation by fluorescence microscopy is that cholesterol depletion does not prevent antigen- dependent aggregation of IgE–Fc ⑀ RI on the cell surface, even though it prevents the co-redistribution of Lyn and inhibits stimulated tyrosine phosphorylation. We previ- ously showed that the cholesterol-binding polyene antibi- otic, filipin, prevents anti-IgE–mediated patching of IgE– Fc ⑀ RI (Feder et al., 1994), indicating that it may prevent aggregation of the receptor necessary to initiate signaling. Unlike M ␤ CD, which extracts cholesterol into a water-sol- uble complex that can be washed away, filipin forms complexes with cholesterol in the membrane that appear to re- strict lateral diffusion of at least some membrane proteins, making this reagent less useful for studying the role of cholesterol in signaling by receptors that must aggregate in response to their ligands to be effective. The rapidity with which M ␤ CD can reduce cell cholesterol by substantial amounts without significantly compromising cell integrity, as evidenced by our degranulation results, and the capac- ity to restore stimulated tyrosine phosphorylation by rein- troduction of cholesterol via M ␤ CD complexes, make this an extremely valuable tool for investigating the role of cholesterol in a wide variety of receptor systems. Recent studies used M ␤ CD to investigate the role of cholesterol in signaling by other receptors. Pike and Miller (1998) showed that cholesterol depletion by M ␤ CD ...

Context 5

... When IgE–Fc ⑀ RI is aggregated by antigen at 22 Њ C for 20 min, small patches of these are formed, and these patches often cluster together on one side of the cell. As see in Fig. 4 c, the concentration of Lyn is enhanced in these regions of patched receptors in the absence of M ␤ CD treatment. For M ␤ CD-treated cells, IgE–Fc ⑀ RI also redistributes into patches after aggregation by antigen (Fig. 4 d, left), indicating that lateral mobility is not im- peded by cholesterol depletion; however, Lyn does not re- distribute with IgE–Fc ⑀ RI under these conditions (Fig. 4 d). As indicated in the first line of Table I, these differences are statistically significant when quantified by cross correlation analysis of multiple cells. Thus, cross-link– dependent interactions between Fc ⑀ RI and Lyn on the cell surface are largely prevented by cholesterol depletion, consistent with the loss of interactions of these proteins with DRMs in the sucrose gradient analyses of lysed cells described above. Fig. 4 e shows that, as previously observed (Pierini et al., 1996), cross-linking of IgE–Fc ⑀ RI at the cell surface results in co-redistribution of the GD 1b ganglioside that is labeled by Cy3-AA4 mAb. For M ␤ CD-treated cells, we find that co-redistribution of this outer leaflet DRM marker with cross-linked IgE–Fc ⑀ RI is reduced compared with control cells but not completely disrupted as was the case for Lyn. Fig. 4 f shows an example of this variability, in which one cell exhibits partial co-redistribution of the labeled gangli- oside, and the other shows a complete lack of co-redistribution with patched IgE–Fc ⑀ RI. Under these conditions, Յ 20% of the cells exhibited detectable co-redistribution of labeled ganglioside, whereas no detectable co-redistribution of Lyn with IgE–Fc ⑀ RI patches was observed. From these results, it appears that the interaction between Lyn and Fc ⑀ RI is more sensitive to cholesterol depletion than is the interaction between the ganglioside and Fc ⑀ RI in the intact cells, although both are substantially prevented. The difference observed may be related to the greater sensitivity of the Lyn–DRM interactions than GD 1b –DRM interactions, as shown in Fig. 3 (see Discussion). As a further control, we compared the distribution of the transferrin receptor (CD71) to FITC-IgE–Fc ⑀ RI cross- linked under the same conditions as above. Previous stud- ies showed that this transmembrane protein does not associate with isolated DRMs (Melkonian et al., 1999), nor does it co-redistribute with other DRM-associated proteins when simultaneously but separately cross-linked on BHK and Jurkat T cells (Harder et al., 1998). As seen in Fig. 4, g and h, TfRs do not co-redistribute with cross- linked IgE–Fc ⑀ RI, and they remain evenly distributed around the periphery of the cell, often in tiny clusters that may reflect interactions with coated pits. Quantitative analysis of the cross correlation of TfR with cross-linked IgE–Fc ⑀ RI show no appreciable colocalization between these molecules whether or not the cells have been depleted of cholesterol (see Table I). These results support the significance of the co-redistribution of Lyn with cross- linked IgE–Fc ⑀ RI described above, as well as its inhibition by cholesterol depletion with M ␤ CD. To investigate the reversibility of cholesterol effects on the functional and structural interactions of Fc ⑀ RI with Lyn, we restored cholesterol levels in M ␤ CD-treated cells by incubating them with cholesterol–M ␤ CD complexes. The efficiency of repletion is dependent upon the incubation period of the cells with the complex, the molar ratio of cholesterol to M ␤ CD, and the final concentration of M ␤ CD (Christian et al., 1997; Sheets, E.D., unpublished results). To optimize repletion, we used several dilutions of cholesterol–M ␤ CD complexes prepared as described in Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control samples) or incubated with M ␤ CD to lower cholesterol levels as described above. During the second step, the cells were incubated for 2 h at 37 Њ C at the indicated dilution of cholesterol/M ␤ CD, 3 mM M ␤ CD only, or buffer only. We found that (a) the cholesterol levels of cells depleted by exposure to M ␤ CD in the first step did not change during the subsequent incubation in the absence of M ␤ CD; (b) the presence of 3 mM of M ␤ CD during the second step also did not cause additional cholesterol depletion, nor did it alter the distribution of IgE–Fc ⑀ RI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3–6 mM M ␤ CD; 8:1, mol/mol M ␤ CD/cholesterol), the cholesterol content of the repleted cells was 3.0–3.5-fold higher than that in the untreated control cells. Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed de- tectably during the depletion/repletion treatments (data not shown). As shown in Fig. 5, repletion of cholesterol in M ␤ CD- treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown, maximal recovery of stimulated tyrosine phosphorylation of Fc ⑀ RI ␤ and other bands was achieved when 1:50 dilution of the preformed 8:1 M ␤ CD/cholesterol complexes was used to give a final concentration of 6 mM M ␤ CD during the repletion step (Fig. 5, lane 10). As seen in Fig. 5, lane 6, cells that had been treated with M ␤ CD alone during both the depletion and repletion steps have no detectable ␤ phosphorylation, and only a very small amount of stimulated tyrosine phosphorylation is seen in the higher molecular weight bands. Similar results to these were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE– Fc ⑀ RI was determined to be 77 Ϯ 4% ( n ϭ 5), and, based upon the proportional relationship between receptor number and ␤ phosphorylation (above), this leads us to expect that optimal restoration of Fc ⑀ RI ␤ phosphorylation should be ف 23% of the stimulated control in Fig. 5, lane 2. The somewhat smaller restoration that is apparent (Fig. 5, lanes 10 and 12) suggests that other factors, such as the loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum restoration achievable (see Discussion). Repletion of cholesterol also results in restoration of cross-link–dependent association of IgE–Fc ⑀ RI with isolated DRMs. When lysates of cholesterol-repleted cells are analyzed on sucrose gradients, cross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭝ ) migrates to the low density sucrose region, whereas uncross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭡ ) is found in the 40% sucrose region, similar to the gradient distributions from control cells (Fig. 3 a, ᭺ and ᭹ ). Cross- linked IgE–Fc ⑀ RI from cholesterol-repleted cells migrate at slightly lower densities in the sucrose gradients than this complex in the control cells, suggesting that the average density of DRMs in repleted cells is lower than in control cells, possibly due to a decrease in the protein/lipid ratio resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates to the low density region of the gradient (fractions 1–6) after cholesterol repletion in cells with both cross-linked and uncross-linked Fc ⑀ RI. These results, in parallel with the restoration of stimulated Fc ⑀ RI tyrosine phosphorylation (Fig. 5), provide strong evidence that cholesterol is important for functional coupling of Fc ⑀ RI with Lyn and for their mutual association with DRMs. Our results demonstrate that cholesterol plays a critical role in the initial step of Fc ⑀ RI signaling: antigen-stimu- lated tyrosine phosphorylation of this receptor by the Src family tyrosine kinase Lyn. In parallel with loss of this stimulated phosphorylation (Fig. 1), reduction of cellular cholesterol by M ␤ CD causes the loss of association of both Lyn and cross-linked Fc ⑀ RI with DRMs isolated after cell lysis by TX-100 (Fig. 3). Restoration of the cholesterol content of the depleted cells using preformed cholesterol–M ␤ CD complexes restores the association of Lyn and cross-linked IgE–Fc ⑀ RI with DRMs (Fig. 3) and also causes partial restoration of antigen-stimulated tyrosine phosphorylation in the cells (Fig. 5). These results support the hypothesis that interactions of cross-linked IgE–Fc ⑀ RI with DRMs are important for the initial coupling of Fc ⑀ RI and Lyn that results in receptor phosphorylation. On the cell surface, the association of Lyn with aggregated IgE– Fc ⑀ RI is lost as the result of cholesterol depletion (Fig. 4), indicating that the interactions detected in isolated DRMs are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked Fc ⑀ RI with Lyn as the basis for association of receptors with DRMs, and they support the view that the L o structure of the plasma membrane is important for Fc ⑀ RI–Lyn interactions. An initially surprising finding in our studies is the appar- ently selective loss of Fc ⑀ RI and outer leaflet plasma membrane components of DRMs due to cholesterol depletion by M ␤ CD. As indicated by Western blot analysis and fluorescence microscopy, there is a smaller loss of Lyn due to cholesterol depletion, and there is no detectable loss of other cellular proteins by silver stain analysis of whole cell lysates (data not shown). The mechanism by which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles containing these components may pinch off from the cells in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by Ilangumaran and Hoessli (1998), a similar preferential release of DRM components by M ␤ CD treatment was characterized in lymphocytes and endothelial ...

Context 6

... is labeled by Cy3-AA4 mAb. For M ␤ CD-treated cells, we find that co-redistribution of this outer leaflet DRM marker with cross-linked IgE–Fc ⑀ RI is reduced compared with control cells but not completely disrupted as was the case for Lyn. Fig. 4 f shows an example of this variability, in which one cell exhibits partial co-redistribution of the labeled gangli- oside, and the other shows a complete lack of co-redistribution with patched IgE–Fc ⑀ RI. Under these conditions, Յ 20% of the cells exhibited detectable co-redistribution of labeled ganglioside, whereas no detectable co-redistribution of Lyn with IgE–Fc ⑀ RI patches was observed. From these results, it appears that the interaction between Lyn and Fc ⑀ RI is more sensitive to cholesterol depletion than is the interaction between the ganglioside and Fc ⑀ RI in the intact cells, although both are substantially prevented. The difference observed may be related to the greater sensitivity of the Lyn–DRM interactions than GD 1b –DRM interactions, as shown in Fig. 3 (see Discussion). As a further control, we compared the distribution of the transferrin receptor (CD71) to FITC-IgE–Fc ⑀ RI cross- linked under the same conditions as above. Previous stud- ies showed that this transmembrane protein does not associate with isolated DRMs (Melkonian et al., 1999), nor does it co-redistribute with other DRM-associated proteins when simultaneously but separately cross-linked on BHK and Jurkat T cells (Harder et al., 1998). As seen in Fig. 4, g and h, TfRs do not co-redistribute with cross- linked IgE–Fc ⑀ RI, and they remain evenly distributed around the periphery of the cell, often in tiny clusters that may reflect interactions with coated pits. Quantitative analysis of the cross correlation of TfR with cross-linked IgE–Fc ⑀ RI show no appreciable colocalization between these molecules whether or not the cells have been depleted of cholesterol (see Table I). These results support the significance of the co-redistribution of Lyn with cross- linked IgE–Fc ⑀ RI described above, as well as its inhibition by cholesterol depletion with M ␤ CD. To investigate the reversibility of cholesterol effects on the functional and structural interactions of Fc ⑀ RI with Lyn, we restored cholesterol levels in M ␤ CD-treated cells by incubating them with cholesterol–M ␤ CD complexes. The efficiency of repletion is dependent upon the incubation period of the cells with the complex, the molar ratio of cholesterol to M ␤ CD, and the final concentration of M ␤ CD (Christian et al., 1997; Sheets, E.D., unpublished results). To optimize repletion, we used several dilutions of cholesterol–M ␤ CD complexes prepared as described in Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control samples) or incubated with M ␤ CD to lower cholesterol levels as described above. During the second step, the cells were incubated for 2 h at 37 Њ C at the indicated dilution of cholesterol/M ␤ CD, 3 mM M ␤ CD only, or buffer only. We found that (a) the cholesterol levels of cells depleted by exposure to M ␤ CD in the first step did not change during the subsequent incubation in the absence of M ␤ CD; (b) the presence of 3 mM of M ␤ CD during the second step also did not cause additional cholesterol depletion, nor did it alter the distribution of IgE–Fc ⑀ RI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3–6 mM M ␤ CD; 8:1, mol/mol M ␤ CD/cholesterol), the cholesterol content of the repleted cells was 3.0–3.5-fold higher than that in the untreated control cells. Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed de- tectably during the depletion/repletion treatments (data not shown). As shown in Fig. 5, repletion of cholesterol in M ␤ CD- treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown, maximal recovery of stimulated tyrosine phosphorylation of Fc ⑀ RI ␤ and other bands was achieved when 1:50 dilution of the preformed 8:1 M ␤ CD/cholesterol complexes was used to give a final concentration of 6 mM M ␤ CD during the repletion step (Fig. 5, lane 10). As seen in Fig. 5, lane 6, cells that had been treated with M ␤ CD alone during both the depletion and repletion steps have no detectable ␤ phosphorylation, and only a very small amount of stimulated tyrosine phosphorylation is seen in the higher molecular weight bands. Similar results to these were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE– Fc ⑀ RI was determined to be 77 Ϯ 4% ( n ϭ 5), and, based upon the proportional relationship between receptor number and ␤ phosphorylation (above), this leads us to expect that optimal restoration of Fc ⑀ RI ␤ phosphorylation should be ف 23% of the stimulated control in Fig. 5, lane 2. The somewhat smaller restoration that is apparent (Fig. 5, lanes 10 and 12) suggests that other factors, such as the loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum restoration achievable (see Discussion). Repletion of cholesterol also results in restoration of cross-link–dependent association of IgE–Fc ⑀ RI with isolated DRMs. When lysates of cholesterol-repleted cells are analyzed on sucrose gradients, cross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭝ ) migrates to the low density sucrose region, whereas uncross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭡ ) is found in the 40% sucrose region, similar to the gradient distributions from control cells (Fig. 3 a, ᭺ and ᭹ ). Cross- linked IgE–Fc ⑀ RI from cholesterol-repleted cells migrate at slightly lower densities in the sucrose gradients than this complex in the control cells, suggesting that the average density of DRMs in repleted cells is lower than in control cells, possibly due to a decrease in the protein/lipid ratio resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates to the low density region of the gradient (fractions 1–6) after cholesterol repletion in cells with both cross-linked and uncross-linked Fc ⑀ RI. These results, in parallel with the restoration of stimulated Fc ⑀ RI tyrosine phosphorylation (Fig. 5), provide strong evidence that cholesterol is important for functional coupling of Fc ⑀ RI with Lyn and for their mutual association with DRMs. Our results demonstrate that cholesterol plays a critical role in the initial step of Fc ⑀ RI signaling: antigen-stimu- lated tyrosine phosphorylation of this receptor by the Src family tyrosine kinase Lyn. In parallel with loss of this stimulated phosphorylation (Fig. 1), reduction of cellular cholesterol by M ␤ CD causes the loss of association of both Lyn and cross-linked Fc ⑀ RI with DRMs isolated after cell lysis by TX-100 (Fig. 3). Restoration of the cholesterol content of the depleted cells using preformed cholesterol–M ␤ CD complexes restores the association of Lyn and cross-linked IgE–Fc ⑀ RI with DRMs (Fig. 3) and also causes partial restoration of antigen-stimulated tyrosine phosphorylation in the cells (Fig. 5). These results support the hypothesis that interactions of cross-linked IgE–Fc ⑀ RI with DRMs are important for the initial coupling of Fc ⑀ RI and Lyn that results in receptor phosphorylation. On the cell surface, the association of Lyn with aggregated IgE– Fc ⑀ RI is lost as the result of cholesterol depletion (Fig. 4), indicating that the interactions detected in isolated DRMs are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked Fc ⑀ RI with Lyn as the basis for association of receptors with DRMs, and they support the view that the L o structure of the plasma membrane is important for Fc ⑀ RI–Lyn interactions. An initially surprising finding in our studies is the appar- ently selective loss of Fc ⑀ RI and outer leaflet plasma membrane components of DRMs due to cholesterol depletion by M ␤ CD. As indicated by Western blot analysis and fluorescence microscopy, there is a smaller loss of Lyn due to cholesterol depletion, and there is no detectable loss of other cellular proteins by silver stain analysis of whole cell lysates (data not shown). The mechanism by which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles containing these components may pinch off from the cells in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by Ilangumaran and Hoessli (1998), a similar preferential release of DRM components by M ␤ CD treatment was characterized in lymphocytes and endothelial cells, and evidence for release of these components in membrane vesicles was described. Our results suggest that Fc ⑀ RI may preferentially associate with DRM components on intact cells even in the absence of receptor cross-linking. Consistent with this, Basciano et al. showed that pre-binding of AA4 mAb or its Fab fragment to the ␣ -galactosyl GD 1b antigen on RBL-2H3 cells can effectively inhibit the subsequent binding of IgE to Fc ⑀ RI (Basciano et al., 1986). By varying the cell surface density of antigen-specific IgE in the range of 30–100%, we show that the loss of Fc ⑀ RI due to cholesterol depletion cannot account for the nearly complete inhibition of Fc ⑀ RI tyrosine phosphorylation that is observed in these cells. Furthermore, the partial restoration of antigen-stimulated tyrosine phosphorylation without an increase in Fc ⑀ RI expression after cholesterol repletion strengthens the evidence that cellular cholesterol critically regulates the coupling of the remaining Fc ⑀ RI and Lyn. An important observation by fluorescence microscopy is that cholesterol depletion does not prevent antigen- dependent aggregation of IgE–Fc ⑀ RI on the cell surface, even ...

Context 7

... surface results in co-redistribution of the GD 1b ganglioside that is labeled by Cy3-AA4 mAb. For M ␤ CD-treated cells, we find that co-redistribution of this outer leaflet DRM marker with cross-linked IgE–Fc ⑀ RI is reduced compared with control cells but not completely disrupted as was the case for Lyn. Fig. 4 f shows an example of this variability, in which one cell exhibits partial co-redistribution of the labeled gangli- oside, and the other shows a complete lack of co-redistribution with patched IgE–Fc ⑀ RI. Under these conditions, Յ 20% of the cells exhibited detectable co-redistribution of labeled ganglioside, whereas no detectable co-redistribution of Lyn with IgE–Fc ⑀ RI patches was observed. From these results, it appears that the interaction between Lyn and Fc ⑀ RI is more sensitive to cholesterol depletion than is the interaction between the ganglioside and Fc ⑀ RI in the intact cells, although both are substantially prevented. The difference observed may be related to the greater sensitivity of the Lyn–DRM interactions than GD 1b –DRM interactions, as shown in Fig. 3 (see Discussion). As a further control, we compared the distribution of the transferrin receptor (CD71) to FITC-IgE–Fc ⑀ RI cross- linked under the same conditions as above. Previous stud- ies showed that this transmembrane protein does not associate with isolated DRMs (Melkonian et al., 1999), nor does it co-redistribute with other DRM-associated proteins when simultaneously but separately cross-linked on BHK and Jurkat T cells (Harder et al., 1998). As seen in Fig. 4, g and h, TfRs do not co-redistribute with cross- linked IgE–Fc ⑀ RI, and they remain evenly distributed around the periphery of the cell, often in tiny clusters that may reflect interactions with coated pits. Quantitative analysis of the cross correlation of TfR with cross-linked IgE–Fc ⑀ RI show no appreciable colocalization between these molecules whether or not the cells have been depleted of cholesterol (see Table I). These results support the significance of the co-redistribution of Lyn with cross- linked IgE–Fc ⑀ RI described above, as well as its inhibition by cholesterol depletion with M ␤ CD. To investigate the reversibility of cholesterol effects on the functional and structural interactions of Fc ⑀ RI with Lyn, we restored cholesterol levels in M ␤ CD-treated cells by incubating them with cholesterol–M ␤ CD complexes. The efficiency of repletion is dependent upon the incubation period of the cells with the complex, the molar ratio of cholesterol to M ␤ CD, and the final concentration of M ␤ CD (Christian et al., 1997; Sheets, E.D., unpublished results). To optimize repletion, we used several dilutions of cholesterol–M ␤ CD complexes prepared as described in Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control samples) or incubated with M ␤ CD to lower cholesterol levels as described above. During the second step, the cells were incubated for 2 h at 37 Њ C at the indicated dilution of cholesterol/M ␤ CD, 3 mM M ␤ CD only, or buffer only. We found that (a) the cholesterol levels of cells depleted by exposure to M ␤ CD in the first step did not change during the subsequent incubation in the absence of M ␤ CD; (b) the presence of 3 mM of M ␤ CD during the second step also did not cause additional cholesterol depletion, nor did it alter the distribution of IgE–Fc ⑀ RI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3–6 mM M ␤ CD; 8:1, mol/mol M ␤ CD/cholesterol), the cholesterol content of the repleted cells was 3.0–3.5-fold higher than that in the untreated control cells. Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed de- tectably during the depletion/repletion treatments (data not shown). As shown in Fig. 5, repletion of cholesterol in M ␤ CD- treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown, maximal recovery of stimulated tyrosine phosphorylation of Fc ⑀ RI ␤ and other bands was achieved when 1:50 dilution of the preformed 8:1 M ␤ CD/cholesterol complexes was used to give a final concentration of 6 mM M ␤ CD during the repletion step (Fig. 5, lane 10). As seen in Fig. 5, lane 6, cells that had been treated with M ␤ CD alone during both the depletion and repletion steps have no detectable ␤ phosphorylation, and only a very small amount of stimulated tyrosine phosphorylation is seen in the higher molecular weight bands. Similar results to these were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE– Fc ⑀ RI was determined to be 77 Ϯ 4% ( n ϭ 5), and, based upon the proportional relationship between receptor number and ␤ phosphorylation (above), this leads us to expect that optimal restoration of Fc ⑀ RI ␤ phosphorylation should be ف 23% of the stimulated control in Fig. 5, lane 2. The somewhat smaller restoration that is apparent (Fig. 5, lanes 10 and 12) suggests that other factors, such as the loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum restoration achievable (see Discussion). Repletion of cholesterol also results in restoration of cross-link–dependent association of IgE–Fc ⑀ RI with isolated DRMs. When lysates of cholesterol-repleted cells are analyzed on sucrose gradients, cross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭝ ) migrates to the low density sucrose region, whereas uncross-linked IgE–Fc ⑀ RI (Fig. 3 a, ᭡ ) is found in the 40% sucrose region, similar to the gradient distributions from control cells (Fig. 3 a, ᭺ and ᭹ ). Cross- linked IgE–Fc ⑀ RI from cholesterol-repleted cells migrate at slightly lower densities in the sucrose gradients than this complex in the control cells, suggesting that the average density of DRMs in repleted cells is lower than in control cells, possibly due to a decrease in the protein/lipid ratio resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates to the low density region of the gradient (fractions 1–6) after cholesterol repletion in cells with both cross-linked and uncross-linked Fc ⑀ RI. These results, in parallel with the restoration of stimulated Fc ⑀ RI tyrosine phosphorylation (Fig. 5), provide strong evidence that cholesterol is important for functional coupling of Fc ⑀ RI with Lyn and for their mutual association with DRMs. Our results demonstrate that cholesterol plays a critical role in the initial step of Fc ⑀ RI signaling: antigen-stimu- lated tyrosine phosphorylation of this receptor by the Src family tyrosine kinase Lyn. In parallel with loss of this stimulated phosphorylation (Fig. 1), reduction of cellular cholesterol by M ␤ CD causes the loss of association of both Lyn and cross-linked Fc ⑀ RI with DRMs isolated after cell lysis by TX-100 (Fig. 3). Restoration of the cholesterol content of the depleted cells using preformed cholesterol–M ␤ CD complexes restores the association of Lyn and cross-linked IgE–Fc ⑀ RI with DRMs (Fig. 3) and also causes partial restoration of antigen-stimulated tyrosine phosphorylation in the cells (Fig. 5). These results support the hypothesis that interactions of cross-linked IgE–Fc ⑀ RI with DRMs are important for the initial coupling of Fc ⑀ RI and Lyn that results in receptor phosphorylation. On the cell surface, the association of Lyn with aggregated IgE– Fc ⑀ RI is lost as the result of cholesterol depletion (Fig. 4), indicating that the interactions detected in isolated DRMs are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked Fc ⑀ RI with Lyn as the basis for association of receptors with DRMs, and they support the view that the L o structure of the plasma membrane is important for Fc ⑀ RI–Lyn interactions. An initially surprising finding in our studies is the appar- ently selective loss of Fc ⑀ RI and outer leaflet plasma membrane components of DRMs due to cholesterol depletion by M ␤ CD. As indicated by Western blot analysis and fluorescence microscopy, there is a smaller loss of Lyn due to cholesterol depletion, and there is no detectable loss of other cellular proteins by silver stain analysis of whole cell lysates (data not shown). The mechanism by which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles containing these components may pinch off from the cells in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by Ilangumaran and Hoessli (1998), a similar preferential release of DRM components by M ␤ CD treatment was characterized in lymphocytes and endothelial cells, and evidence for release of these components in membrane vesicles was described. Our results suggest that Fc ⑀ RI may preferentially associate with DRM components on intact cells even in the absence of receptor cross-linking. Consistent with this, Basciano et al. showed that pre-binding of AA4 mAb or its Fab fragment to the ␣ -galactosyl GD 1b antigen on RBL-2H3 cells can effectively inhibit the subsequent binding of IgE to Fc ⑀ RI (Basciano et al., 1986). By varying the cell surface density of antigen-specific IgE in the range of 30–100%, we show that the loss of Fc ⑀ RI due to cholesterol depletion cannot account for the nearly complete inhibition of Fc ⑀ RI tyrosine phosphorylation that is observed in these cells. Furthermore, the partial restoration of antigen-stimulated tyrosine phosphorylation without an increase in Fc ⑀ RI expression after cholesterol repletion strengthens the evidence that cellular cholesterol critically regulates the coupling of the remaining Fc ⑀ RI and Lyn. An important observation by fluorescence microscopy is that cholesterol depletion does not prevent ...