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Acylation of CD44 and Its Association with Lipid Rafts Are Required for Receptor and Hyaluronan Endocytosis

Thankamony, Sai P., Knudson, Warren·2006·Journal of Biological Chemistry
DOI 10.1074/jbc.m601530200
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FIGURE1 MembranecholesterolisrequiredforCD44-mediatedinternalization of Fl-HA. COS-7 cell transfectants of CD44wt pre-treated without ( A and B ) or with ( C and D ) 10 mM M 𝛃 -CD, were released from monolayers with EDTA and incubated with complete medium containing 15 𝛍 g/ml Fl-HA. To visualize cell surface binding ( A and C ), the cells were incubated for 1 h at 4 °C with Fl-HA, washed, fixed, mounted onto glass slides with DAPI nuclear counterstain and visualized by fluorescence microscopy. To visualize internalized Fl-HA, a corresponding aliquot of cells from each group was incubated with Fl-HA for 3 h at 37 °C followed by extensive trypsinization to remove all extracellularly bound Fl-HA ( B and D ). Shown are digital overlay images of green and blue fluorescence channels. The bars shown in each panel represent 20 𝛍 m.
FIGURE1 MembranecholesterolisrequiredforCD44-mediatedinternalization of Fl-HA. COS-7 cell transfectants of CD44wt pre-treated without ( A and B ) or with ( C and D ) 10 mM M 𝛃 -CD, were released from monolayers with EDTA and incubated with complete medium containing 15 𝛍 g/ml Fl-HA. To visualize cell surface binding ( A and C ), the cells were incubated for 1 h at 4 °C with Fl-HA, washed, fixed, mounted onto glass slides with DAPI nuclear counterstain and visualized by fluorescence microscopy. To visualize internalized Fl-HA, a corresponding aliquot of cells from each group was incubated with Fl-HA for 3 h at 37 °C followed by extensive trypsinization to remove all extracellularly bound Fl-HA ( B and D ). Shown are digital overlay images of green and blue fluorescence channels. The bars shown in each panel represent 20 𝛍 m.CC-BY · From the PDF
FIGURE 2 Association of CD44 with low buoyant density lipid raft membrane fractions. Panel A represents a typical Western blotting profile for CD44 expressed by five different selected clones of Flp-293 cells stably transfected with CD44wt. The clones are labeled as C1 through C5 , whereas E depicts endogenous levels of CD44 expressed by Flp-293 cells. COS-7 transfectants ( B ) and Flp-293 cells ( D ) expressing CD44wt were extracted in the presence of 1% Triton X-100 on ice and subjected to ultracentrifugation on step gradients of iodixanol. Equal volume fractions were collected and analyzed by Western blotting for CD44. The 0-20% iodixanol interface (predominately the 20% fraction) represents the low density, lipid raft fraction. In some instances, cells were cholesterol-depleted by pretreatment with 20 mM M 𝛃 -CDat37 °Cfor50minforCOS-7cells( C ) or 25 min for Flp-293 cells ( E ) prior to ultracentrifugation and Western blotting. The same fractions from the Flp293 stable transfectants shown in D and E were also assayed for the lipid raft marker flotillin-1 using the anti-flotillin-1 antibody, before ( F ) and after ( G ) cholesterol depletion. CD44 was shown to associate with a low buoyant density lipid raft fraction in both COS-7 and Flp-293 cells (verified by the migration of flotillin-1), and this association was dependent on the presence of cholesterol. CD44 was detected using either an anti-CD44 monoclonal antibody, BU-52 ( B and C ) or the epitope-tag anti-V5 monoclonal antibody ( D and E ).
FIGURE 2 Association of CD44 with low buoyant density lipid raft membrane fractions. Panel A represents a typical Western blotting profile for CD44 expressed by five different selected clones of Flp-293 cells stably transfected with CD44wt. The clones are labeled as C1 through C5 , whereas E depicts endogenous levels of CD44 expressed by Flp-293 cells. COS-7 transfectants ( B ) and Flp-293 cells ( D ) expressing CD44wt were extracted in the presence of 1% Triton X-100 on ice and subjected to ultracentrifugation on step gradients of iodixanol. Equal volume fractions were collected and analyzed by Western blotting for CD44. The 0-20% iodixanol interface (predominately the 20% fraction) represents the low density, lipid raft fraction. In some instances, cells were cholesterol-depleted by pretreatment with 20 mM M 𝛃 -CDat37 °Cfor50minforCOS-7cells( C ) or 25 min for Flp-293 cells ( E ) prior to ultracentrifugation and Western blotting. The same fractions from the Flp293 stable transfectants shown in D and E were also assayed for the lipid raft marker flotillin-1 using the anti-flotillin-1 antibody, before ( F ) and after ( G ) cholesterol depletion. CD44 was shown to associate with a low buoyant density lipid raft fraction in both COS-7 and Flp-293 cells (verified by the migration of flotillin-1), and this association was dependent on the presence of cholesterol. CD44 was detected using either an anti-CD44 monoclonal antibody, BU-52 ( B and C ) or the epitope-tag anti-V5 monoclonal antibody ( D and E ).CC-BY · From the PDF
Image 3
CC-BY · From the PDF
FIGURE 3 Palmitoylation is required for CD44 targeting into lipid rafts. 2-BP pre-treatment prevented CD44 association with low buoyant density iodixanol lipid rafts fractions in both COS-7 transient transfectants ( A ) and Flp-293 stable transfectants ( B ) of CD44wt. The palmitoylation mutants of CD44 (pCD44-C286A) expressed in COS-7 transfectants ( C ) and as Flp-293 stable transfectants ( D ) also exhibited significantly reduced association with the low density lipid rafts fraction. Similar results were obtained when the double mutation isoform of CD44, CD44-C286,295A, was expressed in COS-7 transfectants ( E ).
FIGURE 3 Palmitoylation is required for CD44 targeting into lipid rafts. 2-BP pre-treatment prevented CD44 association with low buoyant density iodixanol lipid rafts fractions in both COS-7 transient transfectants ( A ) and Flp-293 stable transfectants ( B ) of CD44wt. The palmitoylation mutants of CD44 (pCD44-C286A) expressed in COS-7 transfectants ( C ) and as Flp-293 stable transfectants ( D ) also exhibited significantly reduced association with the low density lipid rafts fraction. Similar results were obtained when the double mutation isoform of CD44, CD44-C286,295A, was expressed in COS-7 transfectants ( E ).CC-BY · From the PDF
FIGURE 4 [ 3 H]Palmitate is incorporated into CD44 by acylation of membrane-proximalcysteines. A , [ 3 H]palmitateincorporation(250 𝛍 Ci/ml, 2 h at 37 °C) into Flp-293 stable transfectants of CD44wt, pretreated with or without 50 𝛍 M 2-BP ( lanes 1 and 2 , respectively). [ 3 H]Palmitate incorporation into Flp293 stable transfectants of Hyal-2 followed by immunoprecipitation using anti-V5 monoclonal antibody is shown in lane 3 . In B , the extent of [ 3 H]palmitate incorporation in parental Flp-293 cells ( lane 1 ) or Flp-293 cells expressing the wild-type CD44wt ( lane 2 ) were compared with the Flp-293 expressing the cysteine mutants CD44-C286A ( lane 3 ). Lanes 4 and 5 represent a Western blot (detection using an anti-V5 monoclonal antibody) of equivalent aliquots of lysates shown in lanes 2 and 3 , respectively. In C , incorporation of [ 3 H]palmitate into wild-type CD44wt ( lane 1 ), CD44-C286,295A ( lane 2 ), and CD44C286A ( lane 3 ) were compared side-by-side. Lanes 4-6 represent a Western blot (detection using an anti-V5 monoclonal antibody) of equivalent aliquots of lysates shown in lanes 1-3 , respectively. Changes in [ 3 H]palmitate incorporation were quantified by digital scanning of x-ray films, and the resultant data are expressed as the average ± S.D. ( n = 3) change in pixel density relative to control CD44 wild-type incorporation.
FIGURE 4 [ 3 H]Palmitate is incorporated into CD44 by acylation of membrane-proximalcysteines. A , [ 3 H]palmitateincorporation(250 𝛍 Ci/ml, 2 h at 37 °C) into Flp-293 stable transfectants of CD44wt, pretreated with or without 50 𝛍 M 2-BP ( lanes 1 and 2 , respectively). [ 3 H]Palmitate incorporation into Flp293 stable transfectants of Hyal-2 followed by immunoprecipitation using anti-V5 monoclonal antibody is shown in lane 3 . In B , the extent of [ 3 H]palmitate incorporation in parental Flp-293 cells ( lane 1 ) or Flp-293 cells expressing the wild-type CD44wt ( lane 2 ) were compared with the Flp-293 expressing the cysteine mutants CD44-C286A ( lane 3 ). Lanes 4 and 5 represent a Western blot (detection using an anti-V5 monoclonal antibody) of equivalent aliquots of lysates shown in lanes 2 and 3 , respectively. In C , incorporation of [ 3 H]palmitate into wild-type CD44wt ( lane 1 ), CD44-C286,295A ( lane 2 ), and CD44C286A ( lane 3 ) were compared side-by-side. Lanes 4-6 represent a Western blot (detection using an anti-V5 monoclonal antibody) of equivalent aliquots of lysates shown in lanes 1-3 , respectively. Changes in [ 3 H]palmitate incorporation were quantified by digital scanning of x-ray films, and the resultant data are expressed as the average ± S.D. ( n = 3) change in pixel density relative to control CD44 wild-type incorporation.CC-BY · From the PDF
FIGURE 5 Palmitoylation of CD44 is required for HA internalization. COS-7 cells were co-transfected with red fluorescent protein and the wildtype CD44wt ( A and B ) or the CD44-C286S mutant isoform ( C and D ). The transfectants were released from monolayers with EDTA and incubated in complete medium containing 15 𝛍 g/ml Fl-HA. To visualize cell surface binding ( A and C ), the cells were incubated for 1 h at 4 °C with Fl-HA, washed, fixed, mounted onto glass slides with DAPI nuclear counterstain and visualized by fluorescence microscopy ( A and C ). To visualize internalized Fl-HA, a corresponding aliquot of cells from each group was incubated with Fl-HA for 3 h at 37 °C followed by extensive trypsinization to remove all extracellularlybound Fl-HA ( B and D ). Shown are digital overlay images of green and blue fluorescence channels. Insets depict three-channel digital overlays of green , red , and blue fluorescence signals. The bars represent 20 𝛍 m.
FIGURE 5 Palmitoylation of CD44 is required for HA internalization. COS-7 cells were co-transfected with red fluorescent protein and the wildtype CD44wt ( A and B ) or the CD44-C286S mutant isoform ( C and D ). The transfectants were released from monolayers with EDTA and incubated in complete medium containing 15 𝛍 g/ml Fl-HA. To visualize cell surface binding ( A and C ), the cells were incubated for 1 h at 4 °C with Fl-HA, washed, fixed, mounted onto glass slides with DAPI nuclear counterstain and visualized by fluorescence microscopy ( A and C ). To visualize internalized Fl-HA, a corresponding aliquot of cells from each group was incubated with Fl-HA for 3 h at 37 °C followed by extensive trypsinization to remove all extracellularlybound Fl-HA ( B and D ). Shown are digital overlay images of green and blue fluorescence channels. Insets depict three-channel digital overlays of green , red , and blue fluorescence signals. The bars represent 20 𝛍 m.CC-BY · From the PDF
FIGURE 6 Binding and internalization of HA decorated with biotinylated HABP. COS-7 cells were co-transfected with green fluorescent protein and the wild-type CD44H (labeled CD44wt, A and B ), the CD44-C286A mutant isoform ( C and D ), or the CD44-C286,295A double mutant isoform ( E and F ). The transfectants were released from monolayers with EDTA and incubated in complete medium containing 15 𝛍 g/ml unlabeled HA pre-decorated with biotinylated HABP. To visualize cell surface binding ( A , C , and E ), the cells were incubated for 1 h at 4 °C with HA/HABP washed, fixed, mounted onto glass slides with DAPI nuclear counterstain, and visualized by using fluorescence microscopy. To visualize internalized HA/HABP, a corresponding aliquot of cells from each group was incubated with HA/HABP for 3 h at 37 °C followed byextensivetrypsinizationtoremoveallextracellularlyboundHA/HABP( B , D , and F ). Biotinylated HABP was detected using NeutrAvidin-Texas Red. Shown are digital overlay images of red and blue fluorescence channels. Insets depict three-channel digital overlays of green , red , and blue fluorescence signals. The bars represent 20 𝛍 m.
FIGURE 6 Binding and internalization of HA decorated with biotinylated HABP. COS-7 cells were co-transfected with green fluorescent protein and the wild-type CD44H (labeled CD44wt, A and B ), the CD44-C286A mutant isoform ( C and D ), or the CD44-C286,295A double mutant isoform ( E and F ). The transfectants were released from monolayers with EDTA and incubated in complete medium containing 15 𝛍 g/ml unlabeled HA pre-decorated with biotinylated HABP. To visualize cell surface binding ( A , C , and E ), the cells were incubated for 1 h at 4 °C with HA/HABP washed, fixed, mounted onto glass slides with DAPI nuclear counterstain, and visualized by using fluorescence microscopy. To visualize internalized HA/HABP, a corresponding aliquot of cells from each group was incubated with HA/HABP for 3 h at 37 °C followed byextensivetrypsinizationtoremoveallextracellularlyboundHA/HABP( B , D , and F ). Biotinylated HABP was detected using NeutrAvidin-Texas Red. Shown are digital overlay images of red and blue fluorescence channels. Insets depict three-channel digital overlays of green , red , and blue fluorescence signals. The bars represent 20 𝛍 m.CC-BY · From the PDF
Image 8
CC-BY · From the PDF
FIGURE 7 CD44 internalization in COS-7 transfectants is mediated by receptor palmitoylation. COS-7 cells were co-transfected with green fluorescent protein and the wild-type CD44H (labeled CD44wt, A-C ), the CD44-C286A mutant isoform ( D-F ), or the CD44-C286,295A double mutant isoform ( G-I ). The transfectants were then preincubated without ( A , D , and G ) or with 100 𝛍 g/ml cycloheximide for 3 h at 37 °C ( B , C , E , F , H , and I ) as labeled. To detect intracellular staining, the cells were co-incubated with cycloheximide and chloroquine; the latter was used to inhibit lysosomal degradation and enhance visualization of internalized receptor ( C , F , and I ). Cell surface CD44 was visualized by immunostaining of fixed cells with the anti-CD44 monoclonal antibody, BU-52, and anti-mouse cy-3-conjugated secondary antibody ( red fluorescence; A , B , D , E , G , and H ). To detect internalized CD44 ( C , F , and I ), cells were trypsinized, fixed, and permeabilized prior to immunostaining with the BU-52 antibody. Shown are digital overlay images of red and blue fluorescence channels. Insets depict three-channel digital overlays of green , red , and blue fluorescence signals. The bars represent 20 𝛍 m.
FIGURE 7 CD44 internalization in COS-7 transfectants is mediated by receptor palmitoylation. COS-7 cells were co-transfected with green fluorescent protein and the wild-type CD44H (labeled CD44wt, A-C ), the CD44-C286A mutant isoform ( D-F ), or the CD44-C286,295A double mutant isoform ( G-I ). The transfectants were then preincubated without ( A , D , and G ) or with 100 𝛍 g/ml cycloheximide for 3 h at 37 °C ( B , C , E , F , H , and I ) as labeled. To detect intracellular staining, the cells were co-incubated with cycloheximide and chloroquine; the latter was used to inhibit lysosomal degradation and enhance visualization of internalized receptor ( C , F , and I ). Cell surface CD44 was visualized by immunostaining of fixed cells with the anti-CD44 monoclonal antibody, BU-52, and anti-mouse cy-3-conjugated secondary antibody ( red fluorescence; A , B , D , E , G , and H ). To detect internalized CD44 ( C , F , and I ), cells were trypsinized, fixed, and permeabilized prior to immunostaining with the BU-52 antibody. Shown are digital overlay images of red and blue fluorescence channels. Insets depict three-channel digital overlays of green , red , and blue fluorescence signals. The bars represent 20 𝛍 m.CC-BY · From the PDF
FIGURE8 CD44internalization is dependent on receptor palmitoylation. The two channel fluorescence overlay images of cells ( A and B ) represent another illustration of the loss of CD44 expression in COS-7 transfectants of CD44wt at time zero ( A ) as compared with 3 h following pre-treatment with cycloheximide ( B ). As in Fig. 7, the red fluorescence represents immunostaining of the cells with anti-CD44 monoclonal antibody, BU-52, followed by anti-mouse Cy3-conjugated secondary antibody. C depicts CD44 Western blotting of cell lysates prepared from COS-7 transfectants at time zero ( lanes 1 , 3 , and 5 ) or after 6 h of treatment with cycloheximide ( lanes 2 , 4 , and 6 ). Lysates of wild-type CD44 (CD44wt; lanes 1 and 2 ), CD44-C286A ( lanes 3 and 4 ), or CD44-C286,295A ( lanes 5 and 6 ) were examined. D represents a CD44 Western blot of another experiment that depicts transfectants of CD44H ( lanes 1-3 ) or CD44-C286,295A ( lanes 4 and 5 ) at time zero ( lanes 1 and 4 ), treated with cycloheximide for 3 h ( lanes 2 and 5 ) or treated with cycloheximide and chloroquine for 3 h ( lane 3 ). Changes in band intensity were quantified by digital scanning of x-ray films, and the resultant data are expressed as the average ± S.D. ( n = 3) change in pixel density relative to control CD44 wild-type expression.
FIGURE8 CD44internalization is dependent on receptor palmitoylation. The two channel fluorescence overlay images of cells ( A and B ) represent another illustration of the loss of CD44 expression in COS-7 transfectants of CD44wt at time zero ( A ) as compared with 3 h following pre-treatment with cycloheximide ( B ). As in Fig. 7, the red fluorescence represents immunostaining of the cells with anti-CD44 monoclonal antibody, BU-52, followed by anti-mouse Cy3-conjugated secondary antibody. C depicts CD44 Western blotting of cell lysates prepared from COS-7 transfectants at time zero ( lanes 1 , 3 , and 5 ) or after 6 h of treatment with cycloheximide ( lanes 2 , 4 , and 6 ). Lysates of wild-type CD44 (CD44wt; lanes 1 and 2 ), CD44-C286A ( lanes 3 and 4 ), or CD44-C286,295A ( lanes 5 and 6 ) were examined. D represents a CD44 Western blot of another experiment that depicts transfectants of CD44H ( lanes 1-3 ) or CD44-C286,295A ( lanes 4 and 5 ) at time zero ( lanes 1 and 4 ), treated with cycloheximide for 3 h ( lanes 2 and 5 ) or treated with cycloheximide and chloroquine for 3 h ( lane 3 ). Changes in band intensity were quantified by digital scanning of x-ray films, and the resultant data are expressed as the average ± S.D. ( n = 3) change in pixel density relative to control CD44 wild-type expression.CC-BY · From the PDF

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Authors
Thankamony, S. P., & Knudson, W.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2006-11
Volume
281
Issue
45
Pages
34601-34609
ISSN
0021-9258
DOI
10.1074/jbc.m601530200
Cited by
110
References
0
Language
en
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