HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 22, 19946-19951, May 31, 2002

This Article Abstract Full Text (PDF) Supplemental Data An addition or correction has been published All Versions of this Article: 277/22/19946    most recent M200841200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tkachenko, E. Articles by Simons, M. Search for Related Content PubMed PubMed Citation Articles by Tkachenko, E. Articles by Simons, M. Social Bookmarking                      What's this?

Clustering Induces Redistribution of Syndecan-4 Core Protein into Raft Membrane Domains^*

Eugene Tkachenko and Michael Simons

From the Angiogenesis Research Center and Section of Cardiology, Department of Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756

Received for publication, January 26, 2002, and in revised form, March 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Syndecan-4 is a heparan sulfate-carrying core protein that has been directly implicated in fibroblast growth factor 2 (FGF2) signaling. Recent studies have suggested that many signaling proteins localize to the raft compartment of the plasma cell membrane. To establish whether syndecan-4 is present in the raft compartment, we have studied the distribution of the core protein and an Fc receptor (FcR)-syndecan-4 chimera prior to and following clustering with FGF2 or antibodies. Whereas unclustered syndecan-4 was present predominantly in the non-raft membrane compartment, clustering induced extensive syndecan-4 redistribution to the rafts as demonstrated by the sucrose gradient centrifugation and life confocal microscopy. Although syndecan-4 and caveolin-1 moved in tandem, syndecan-4 was not present in caveolae, a major subset of raft compartments. We conclude that syndecan-4 clustering induces its redistribution to the non-caveolae raft compartment. This process may play an important role in syndecan-4-mediation of FGF2 signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Syndecans, a four-member family of transmembrane proteoglycan core proteins, are found in a wide spectrum of cells and engage in a variety of interactions including binding growth factors, growth factor receptors, matrix proteins such as fibronectin and vitronectin, lipids, and others with biologically active molecules (1, 2). All syndecans carry both heparan sulfate and chondroitin sulfate chains on their extracellular domain and engage in PDZ (Postsynaptic density 95, Disk large, Zona occludens-1)-dependent interaction via conserved intracellular domains (1, 2). Syndecan-4 is a unique member of the syndecan core protein family. Unlike other syndecans, it possesses a phosphoinositol 4,5-bisphosphate binding site in its cytoplasmic tail that allows it to bind and activate protein kinase C (3, 4). Syndecan-4 is found on endothelial cells and is directly involved in the regulation of FGF2-induced cell growth and migration (5).

Although previous studies have shown that syndecan-4 is predominantly found on the basolateral plasma cell membrane and focal adhesions (6, 7), little information is available regarding its association with various membrane subdomains. Recent studies have suggested that rafts, lipid-ordered microdomains enriched by cholesterol and sphingolipids, act as platforms for conducting a variety of cellular functions such as vesicular trafficking and signal transduction (8, 9). Therefore, we have conducted this study to determine whether activation of syndecan-4 signaling initiated by its oligomerization on the plasma cell membrane leads to its appearance in the membrane rafts.

Several protein families have been reported to modify lipid rafts structurally and functionally. These include integral membrane proteins such as caveolins and flotillins, exoplasmic glycosylphosphatidylinositol (GPI)¹-linked proteins such as Thy-1 and alkaline phosphatase, and receptor tyrosine kinases among others (9). Caveolin integration into the microenvironment of a lipid raft leads to raft invagination and formation of caveolae (9-11). Caveolae are formed in many cell types including endothelial cells (12), and they may play a role in modulation of cell signaling. One hypothesis suggests that interactions of caveolins with signaling molecules regulate their activation status.

Because FGF2-induced syndecan-4-dependent signaling requires the presence of heparan sulfate chains on core protein extracellular domain (13), it is reasonable to hypothesize that FGF2 induces aggregation of syndecan-4 complexes in the plasma cell membrane. However, because FGF2 induces a number of other signaling events that may affect syndecan-4 movement in the plasma cell membrane in manner independent of oligomerization of its extracellular domains, we linked the extracellular domain of the human Fc receptor (FcR)-Ia to the transmembrane/cytoplasmic domains of syndecan-4. The resultant chimera can then be aggregated with an immunoglobulin, mimicking FGF2-induced syndecan-4 oligomerization. We found that aggregation of syndecan-4 cores leads to a shift of syndecan-4 complexes from the non-raft to raft microdomains of the plasma cell membrane. Although syndecan-4 motion was synchronous with movements of caveolin-1, syndecan-4 was not present in the caveolae portion of the plasma membrane rafts.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Antibodies and Reagents-- Polyclonal rabbit antisera against cytoplasmic and extracellular domains of syndecan-4 were described elsewhere (14). Polyclonal chicken IgY against extracellular domains of syndecan-4 was produced (Aves Laboratories) using the same peptide as previously used for generation in rabbits (14). Anti-FcR (CD64) monoclonal antibody was purchased from Abcam (Cambridge, United Kingdom). Cy-3 and biotin-SP-conjugated non-immune human IgG, Cy-3-conjugated goat anti-human F(ab')₂ fragment, Cy-5-conjugated streptavidin, and goat anti-chicken F(ab')₂ fragment were purchased from Jackson ImmunoResearch. Secondary antibodies conjugated to Alexa-594 and a nuclear stain ToPro3 were from Molecular Probes. Alexa-488-conjugated inactive variant of the protein proaerolysin (FLAER) was purchased from Protox Biotech (Victoria, British Columbia, Canada). Secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Vector Laboratories, and immobilized streptavidin and HRP-conjugated streptavidin were from Pierce. HRP-conjugated cholera toxin  subunit was purchased from Sigma.

cDNA Constructs and Transfection-- Fc receptor-syndecan-4 chimera (FcR-S4) was constructed by linking the ectoplasmic domain (amino acids 1-288) of human Fc receptor Ia (CD64) cDNA and the transmembrane and cytoplasmic domains of rat syndecan-4 (amino acids 150-202). The chimera and the full-length syndecan-4 construct were inserted into pCR 3.1-Uni expression vector (Invitrogen). Caveolin- 1-EGFP cDNA construct (15) was a gift of Dr. L. Pelkmans (Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland).

RFPEC were cultured in DMEM medium (Invitrogen) as described previously (16). Stable expression of FcR-S4 chimera was achieved by transfecting the cDNA construct into wild-type RFPEC using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. Cells were selected for neomycin resistance (0.4 mg/ml Geneticin, Invitrogen), and pooled populations were used for all studies. To enrich each pool with cells having high expression levels of FcR-S4 protein, cells were subjected to two rounds of fluorescent-activated sorting (see below) to select cells in the top 10% of the population.

Transient expression of caveolin-1-EGFP and pEGFP-N1 (CLONTECH) in RFPEC was achieved by transfection using LipofectAMINE 2000. Cells were used within 48-72 h after transfection.

Fluorescence-assisted Cell Sorting-- Cells were dissociated from plates using non-enzymatic solution (Sigma) and labeled for 20 min with fluorescein isothiocyanate-conjugated human non-immune IgG (0.1 µg/ml, Rockland) in Dulbecco's phosphate-buffered saline with 1% bovine serum albumin (BSA), thus specifically detecting the transfected Fc receptor that is not endogenously expressed in endothelial cells. To detect endogenous syndecan-4, cells were incubated with 1 µg/ml chicken antibody against extracellular domain of syndecan-4 for 20 min at room temperature, washed twice in 1% BSA-DMEM, and then incubated with a secondary antibody (Cy-5-conjugated F(ab')₂ fragment, 1 µg/ml) for 20 min at room temperature followed by another wash with 1% BSA-DMEM. Cell sorting was carried out on a MoFlo sorter (cytomation) with a FACScan (BD PharMingen) and analyzed using WinMDI version 2.8 (Scripps Institute, La Jolla, CA) software.

Cell Surface Biotinylation-- The cells were washed twice with ice-cold DMEM and incubated with 0.5 mg/ml sulfo-N-hydroxysuccinimide ester-long chain biotin (Pierce) in phosphate-buffered saline, pH 7.4, for 15 min at 4 °C. The biotinylation reaction was quenched with ice-cold 1% BSA-DMEM.

Syndecan Clustering-- For antibody-clustering studies, RFPEC-expressing FcR-S4 construct were grown overnight in 10% FBS-DMEM. The cells were washed twice in DMEM and then incubated with human non-immune IgG (1 µg/ml) for 15 min in 1% BSA-DMEM at 37 °C. The cells were then washed again twice with DMEM and incubated for another 15 min at 37 °C with anti-human F(ab')₂ (2 µg/ml) in 1% BSA-DMEM.

For FGF2 clustering, RFPEC cultured in 10% FBS-DMEM were washed twice with DMEM and then incubated with 25 ng/ml FGF2 (Chiron Corp, Sunnyvale, CA) in 1% BSA-DMEM for 15 min at 37 °C.

Isolation of Lipid Raft Membrane Fractions-- Raft membrane microdomains from cell surface-biotinylated RFPEC were isolated using flotation on discontinuous sucrose gradients as described previously (17). Adherent cells in confluent 15-cm dish were washed with ice-cold phosphate-buffered saline and lysed for 30 min on ice in 1% Triton X-100 in MNE buffer (150 mM NaCl, 2 mM EDTA, 25 mM, pH 6.5) containing a protease inhibitor mixture (Roche Diagnostics). The lysis solution was homogenized, and nuclei and cellular debris were pelleted by centrifugation at 10,000 × g for 30 min. For sucrose-gradient centrifugation, 1 ml of the cleared supernatant was mixed with 1 ml of 85% sucrose in MNE buffer and transferred to the bottom of a centrifuge tube. The diluted lysate was overlaid with 2 ml of 30% sucrose and 1 ml of 5% sucrose in MNE buffer. The samples were centrifuged in an SW55 rotor at 200,000 × g at 4 °C for 16 h. Seven fractions of 700 µl were collected from the top of the gradient and studied using immunoblotting (see below).

Detergent extraction of lipid rafts was carried as described previously (18). Cells were grown to confluence in a 6-well plate, rinsed with 1 ml of ice-cold TNE (Tris-NaCl-EDTA) buffer (25 mM Tris-Cl, pH 7.4, 150 mM NaCl, and 5 mM EDTA), placed on ice, and then lysed by the addition of 1% Triton X-100 and protease inhibitor mixture in TNE buffer for 20 min. Lysates were scraped and centrifuged for 5 min at 4 °C, and the supernatant was transferred to the fresh tube. To recover membrane rafts proteins, Triton X-100 pellet was resuspended in 100 µl of pellet lysis buffer (50 mM Tris-HCl, pH 8.8, 5 mM EDTA, and 1% (w/v) SDS). The mixture was diluted with 1 ml of TNE-1% Triton X-100 and centrifuged for 2 min at 4 °C. The supernatant was then analyzed by immunoprecipitation, SDS-PAGE, and immunoblotting. Glycosaminoglycan chains were digested as described previously (4).

Plasma membrane cholesterol depletion was accomplished by pretreatment of cultured cells with -cyclodextrin (10 µM), which removes unesterified cholesterol from the cell membrane (19) for 30 min at 37 °C immediately followed by chilling the cells to 4 °C.

Immunoprecipitation, Immunoblotting, and Dot-blotting-- Immunoprecipitation of syndecan-4 from cell lysates was carried out by incubation of 500 µl of total cell lysates with 10 µl of anti-syndecan-4 cytoplasmic tail antiserum in the presence of protein G/protein A-agarose (30 µl). Prior to immunoblotting, glycosaminoglycan chains were digested as described previously (4). The immunoprecipitated material was then subjected to SDS-PAGE and transferred to the polyvinylidene difluoride membrane (Millipore). Syndecan-4 detection was performed using HRP-conjugated streptavidin (0.2 µg/ml) for cell surface-biotinylated samples or by anti-syndecan-4 antiserum (1 µl/ml) followed by incubation with a secondary HRP-conjugated goat antibody specific for the primary anti-syndecan-4 antibody.

Immunoprecipitation of syndecan-4 from sucrose gradient fractions was performed in the similar manner. The precipitated material was then Dot-blotted on the nitrocellulose membrane (Schleicher & Schuell), and syndecan-4 was detected by HRP-conjugated streptavidin.

For GM1 sphingolipid detection in sucrose gradient fractions, 100 µl of each fraction were applied to the nitrocellulose membrane, and GM1 was then detected using HRP-conjugated cholera toxin subunit  (0.2 µg/ml). GM1 detection in Triton X-100-soluble and insoluble fractions was carried out by blotting 20 or 200 µl of each fraction on the nitrocellulose filter, which was then probed with HRP-conjugated cholera toxin subunit  (0.2 µg/ml). All blots were developed using enhanced SuperSignal Wet Pico chemiluminescent substrate (Pierce).

Live Fluorescent Microscopy-- For live fluorescent microscopy, cells were grown on gelatin 0.1% phosphate-buffered saline-coated coverslips (Fisher) to confluence. Staining for FcR-S4 was done using biotinylated non-immune IgG, and the biotin label was then visualized by incubation with Cy-5-conjugated streptavidin (5 µg/ml) at 37 °C for 15 min. GPI-anchored proteins were detected by cell incubation with 10⁸ M FLAER at 37 °C for 1 h. FLAER binds selectively to GPI anchors (20).

Coverslips were removed from the staining solution and mounted on a microscope slide. All microscopy imaging was done using Bio-Rad MRC-1024 krypton/argon laser confocal system microscope with ×63 lens by Zeiss. Time-lapse microscopy was carried out at 20 °C. Image analysis was done using Adobe Photoshop 6.0 (Adobe Systems Inc.) and ImageJ software (NIH).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To study the distribution of syndecan-4 core protein in the plasma cell membrane and examine the effect of ligand clustering on its spatial localization, we linked the cytoplasmic and the transmembrane domains of syndecan-4 to the extracellular domain of the Fc receptor. To demonstrate the expression of the FcR-S4 chimera on the cell surface, FcR-S4 construct and empty vector-transfected cells were subjected to fluorescence-assisted cell sorting using fluorescein isothiocyanate-labeled non-immune IgG that specifically recognizes Fc receptor and anti-syndecan-4 ectoplasmic domain antibodies. FcR expression was noted only on the FcR-S4 construct-transfected cells (Fig. 1A). Furthermore, the expression of the native syndecan-4 was not affected by FcR-S4 expression (Fig. 1A). To demonstrate that the cytoplasmic tails of FcR-S4 chimeras interact with native syndecan-4 proteins, RFPEC cells expressing FcR-S4 construct were decorated with non-immune IgG. The clustering of FcR-syndecan-4 complexes was then performed with the anti-IgG antibody. Clustered and unclustered RFPEC were then lysed and subjected to immunoprecipitation with the antibody against the ectoplasmic domain of syndecan-4 followed by Western blotting with an anti-FcR (CD64) antibody. Both clustered and unclustered cells demonstrated the presence of FcR-S4-syndecan-4 complexes (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   FcR-syndecan-4 chimera interaction with endogenous syndecan-4 on the cell surface. A, flow cytometry analysis of FcR-S4 and native syndecan-4 expression in RFPEC. Fluorescent intensity of antibody staining for the FcR and native syndecan-4 in cells transfected with the FcR-S4 construct (red) or vector control (green). Note the absence of signal for the FcR (non-immune IgG) in the vector-transfected cells (green). The expression of the native syndecan-4 is the same in vector-transfected (orange) and FcR-S4-transfected (blue) cells. B, FcR-S4 forms complexes with native syndecan-4. Total cell lysates from cells transfected with FcR-S4 construct prior to () or after (+) antibody clustering of FcR-S4 chimeras were subjected to immunoprecipitation with the anti-ectoplasmic domain syndecan-4 antibody followed by Western blotting of the precipitate with anti-FcR (CD64) antibody. Note the presence of bands corresponding to FcR-S4-syndecan-4 monomer and dimer.

The distribution of syndecan-4 in the plasma cell membrane was examined by subjecting whole cells lysates of RFPEC-expressing FcR-S4 construct to sucrose density gradient centrifugation. Blotting of the syndecan-4 antibody-immunoprecipitated material from various sucrose gradient fractions demonstrated the core protein presence in both heavy (40% sucrose) and light (10-15% sucrose) fractions (Fig. 2A, right panel). The lighter fractions contain plasma membrane raft proteins as shown by blotting with HRP-conjugated cholera toxin subunit , which specifically binds to the raft marker GM1 (Fig. 2A, left panel). In the absence of antibody clustering, most of syndecan-4 appeared in the non-raft membrane fractions. Antibody clustering of FcR-S4 chimeras induced a pronounced shift toward the raft-containing fractions (Fig. 2A, right panel). The specificity of HRP-conjugated cholera toxin subunit -GM1 binding for the detection of raft fractions of the membrane was tested by Dot-blot analysis of Triton X-100-soluble and insoluble portion of the plasma cell membrane (Fig. 2B). As expected, only the Triton X-100-insoluble fraction was labeled with the cholera toxin.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Clustering induces syndecan-4 redistribution into the lipid rafts. A, sucrose gradient analysis. Raft membrane microdomains from cell surface biotinylated RFPEC were isolated using flotation on discontinuous sucrose gradients. Gradient fractions containing rafts were detected using HRP-conjugated cholera toxin subunit  that identifies the presence of the raft marker GM1 (left panel). The distribution of FcR-S4 in the gradient fractions was determined by immunoprecipitation of each fraction with the anti-syndecan-4 cytoplasmic domain antibody. The precipitated material was then Dot-blotted, and syndecan-4 was detected by HRP-conjugated streptavidin (right panel). Note that prior to clustering most of syndecan-4 is present in the non-raft sucrose fractions with a shift toward the raft fraction following antibody clustering of FcR-S4 constructs. B, raft microdomains are present in Triton X-100-insoluble fractions. To demonstrate the presence of raft microdomains in the Triton X-100-insoluble fraction, RFPEC was subjected to lysis in Triton X-100 at 4 °C as described under "Materials and Methods." 20 or 200 µl of each fraction were blotted on the nitrocellulose filter, which was then probed with the HRP-conjugated cholera toxin subunit . Note the presence of GM1 signal only in Triton X-100-insoluble fractions. C, antibody clustering induces the shift of FcR-S4 chimeras from non-raft to raft membrane microdomains. RFPEC-expressing FcR-syndecan-4 decorated with non-immune IgG were subjected to cold Triton X-100 extraction followed by immunoprecipitation with antibodies against the cytoplasmic domain of syndecan-4 and Western blotting with HRP-streptavidin. Prior to clustering (), syndecan-4 is predominantly found in the soluble (non-raft) fraction (lanes 1 and 2), whereas the antibody clustering of FcR-syndecan-4 chimeras (+) induces pronounced shift toward the insoluble (raft) fraction (lanes 3 and 4) lipid rafts. Cholesterol depletion of the plasma cell membrane with -cyclodextrin led to a pronounced reduction in clustering the induced shift of syndecan-4 toward the raft fraction (lanes 5 and 6). D, FGF2 treatment shifts native syndecan-4 to the raft microdomains. RFPEC treated with FGF-2 were cell surface-biotinylated and then subjected to Triton X-100 extraction at 4 °C followed by immunoprecipitation with antibodies against the cytoplasmic domain of syndecan-4 and Western blotting with HRP-streptavidin. Note paucity of syndecan-4 protein in the raft fraction prior to FGF2-induced clustering (lanes 1 and 2) and a pronounced shift toward the raft fraction following FGF2 treatment (lanes 3 and 4).

To further explore the relationship between clustering and syndecan-4 redistribution into the raft compartment, we studied syndecan-4 appearance in the Triton X-100-insoluble (raft) and soluble (non-raft) fractions of plasma cell membranes. Equal numbers of FcR-S4 expressing cells were subjected to lysis in Triton X-100 as described under "Materials and Methods." The Triton X-100-soluble and insoluble fractions were then immunoprecipitated with anti-syndecan-4 antibody, separated on SDS-PAGE, and subjected to Western blotting with HRP-streptavidin. Prior to antibody clustering of FcR-S4 chimeras, essentially all of syndecan-4 was in the Triton X-100-soluble fraction (Fig. 2C). Antibody clustering induced redistribution of ~50% cell surface syndecan-4 to the Triton X-100-insoluble fraction. Because the formation of membrane rafts depends on high local cholesterol ester content, we disrupted raft formation by depleting membrane cholesterol by treating cells with -cyclodextrin. Such cholesterol depletion almost fully prevented clustering-induced syndecan-4 redistribution into the rafts compartment (Fig. 2C).

Because clustering of the native syndecan-4 with its natural ligands such as FGF2 may potentially have a different effect on its plasma membrane dispersal than antibody clustering of FcR-S4 chimeras, we have examined the effect of FGF2 treatment on syndecan-4 distribution in RFPEC cells. Similar to the FcR-S4 chimera antibody clustering studies, FGF2 induced a significant redistribution of syndecan-4 from Triton X-100-soluble to insoluble fraction (Fig. 2D).

To confirm localization of clustered syndecan-4 to plasma membrane rafts, we used vital confocal microscopy with anti-syndecan-4 antibody and the GPI-anchor marker FLAER in RFPEC. Following antibody clustering of FcR-S4 chimeras, FLAER imaging demonstrated prominent raft clusters on the apical plasma cell membrane (Fig. 3, top left panel). Staining with a labeled non-immune IgG that detects FcR-S4 chimeras demonstrated equally prominent FcR-S4 clusters on the apical plasma cell membrane (Fig. 3, top middle panel). The merged image demonstrates that essentially all FcR-S4 chimeras overlapped with FLAER labeling (Fig. 3, top right panel), consistent with syndecan-4 presence in the FLAER-labeled rafts. The specificity of staining was confirmed by FLAER and non-immune IgG labeling of the vector-transfected RFPEC (Fig. 3, bottom panels) that demonstrated the absence of FcR-S4 signal.

View larger version (99K): [in this window] [in a new window]   Fig. 3.   FcR-Syndecan-4 co-localizes with the GPI-anchored protein marker FLAER in RFPEC. Confocal images of live RFPEC transfected with the FcR-S4 construct (top) or vector control (bottom). Prior to immunolabeling, cells were subjected to antibody clustering of FcR-S4 chimeras as described under "Materials and Methods." Labeling with the GPI-anchored proteins marker FLAER (green) is shown in the left panel, whereas labeling of FcR-S4 using biotinylated non-immune IgG visualized by Cy-5-conjugated streptavidin is shown in the middle panel. The merged image is shown in the right panel. Note a virtually complete overlap of FcR-S4 signal with the FLAER signal. Because there is a large excess of GPI proteins compared with FcR-S4 chimeras, not all FLAER signal demonstrates co-localization with the FcR-S4 signal. Note the absence of FcR-S4 signal in vector-transfected cells (bottom panels).

Caveolae constitute a distinct subpopulation of cholesterol-rich rafts. To see whether syndecan-4 is present in caveolae, we generated RFPEC cells expressing EGFP-tagged caveolin-1. Live confocal microscopy showed no co-localization between syndecan-4 and caveolin-1 (Fig. 4A) and coordinated the movement of the two proteins (movie file). To explore the effect of syndecan-4 clustering on its association with caveolae, we used time-lapse microscopy to visualize syndecan-4 movement in caveolin-1-EGFP-expressing RFPEC cells. The reconstruction of time stacks of syndecan-4-caveolin-1 images in the x and y plane demonstrated that although both proteins moved together, they did not localize to the same cellular compartment (Fig. 4B). Western blots with an anti-caveolin-1 antibody failed to detect its presence among proteins present in the syndecan-4 immunoprecipitations (data not shown). To control for the effect of EGFP expression on the cellular distribution of caveolin-1-EGFP chimeric protein, RFPEC were transiently transfected with an EGFP construct. A visualization of EGFP demonstrated diffuse distribution of the protein in the cytoplasm (Fig. 4C), quite distinct from the appearance of caveolin-1-EGFP protein. Syndecan-4 staining is present along the cell border (Fig. 4C).

View larger version (62K): [in this window] [in a new window]   Fig. 4.   FcR-Syndecan-4 associates with caveolae. A, syndecan-4 association with caveolae. Differential interference contrast (DIC) image of live RFPEC co-transfected with caveolin-1-EGFP and FcR-S4 constructs is overlaid by images of caveolin-1-EGFP (green) and clustered FcR-S4 (red). Note a lack of overlap of caveolin-1-EGFP and FcR-S4 signals. Inset depicts digitally magnified view of the indicated region. An associated movie file demonstrates co-movement of syndecan-4 and caveolin-1. Time-lapse series of 48 images from the inset were acquired with a 2.5-s interval. Note the association but not an overlap of syndecan-4 and caveolin-1 signals. B, spatial analysis of caveolin-syndecan-4 movement. Movement of the caveolin-1-EGFP (green) and syndecan (red) signals were tracked over time in x and y planes. The graphs display the relative motion along the y (top) and x (bottom) axis over time. Note the tandem movement but lack of overlap of the two proteins. C, EGFP expression in RFPEC. To control for the effect of EGFP expression on the behavior of caveolin-1-EGFP construct, RFPEC cells were transfected with EGFP vector. The DIC image of live RFPEC-expressing EGFP is overlaid with the EGFP (green) and FcR-S4 (red) signals. Note a diffuse cytoplasmic distribution of EGFP that is quite distinct from the appearance of caveolin-1-EGFP. Syndecan-4 staining (red) is present along the cell border.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Whereas syndecans have largely been considered structural proteins, recent data suggest that they play a variety of roles including lipoprotein uptake (21), cell adhesion (6), and regulation of FGF2 signaling (5, 22). However, the molecular events involved in these activities are not well understood. Previous studies have shown that in Chinese hamster ovary cells, syndecan-1 is found upon clustering in the Triton X-100-insoluble regions of the membrane (23) and that this event may play a role in syndecan-1-mediated lipid endocytosis (21, 24). Although syndecan-1 and syndecan-4 share significant homologies in the transmembrane and cytoplasmic regions domains, which are the regions thought to play a role in clustering-induced aggregation in membrane rafts, there are also significant differences. Syndecan-4 is a unique member of the syndecan core protein family because of its ability to engage in FGF2 signal transduction. Furthermore, no data have previously been available regarding the effect of FGF2 binding on syndecan-4 clustering and the occurrence of this event in polarized endothelial cells.

Because FGF2 treatment induces a number of cellular events that can potentially affect membrane syndecan-4 trafficking, we employed FcR-syndecan-4 chimeras to study the effect of syndecan clustering on its localization in the plasma cell membrane. The use of extracellular domain of Fc receptor is particularly convenient, because Fc receptor is not expressed in endothelial cells and non-immune IgG can be used to decorate cells expressing Fc receptor. The chimera protein behaves in a mode analogous to the native syndecan-4. It is present on the cell surface, it forms complexes with the native syndecan-4, and its expression does not affect the expression of the native protein. Therefore, FcR-S4 clustering behavior is representative of ligand clustering of the native syndecan-4.

We find that in quiescent endothelial cells, FcR-S4 chimeras as well as native syndecan-4 are present mostly in the non-raft fraction of the membrane and that the aggregation of FcR-S4 chimeras with antibodies or syndecan-4 with FGF2 leads to a rapid redistribution of the proteins to the membrane rafts. Several independent pieces of data are consistent with this interpretation including sucrose gradients, Western blotting of various membrane fractions, and life confocal microscopy.

Two principle raft subdomains are caveolae-containing and caveolae-free rafts. Interestingly, syndecan-4 did not co-localize with caveolin-1, suggesting that it was not present in the caveolae but rather in the non-caveolae rafts. The absence of caveolin-1 in the syndecan-4-immunoprecipitated material further argues against direct interaction between these proteins. At the same time, the clustering of FcR-S4 chimeras led to close apposition, but apparently not the binding, of syndecan-4 and caveolae and a complex-coordinated movement of both proteins.

Although we have not examined which portion of the syndecan-4 molecule mediates its association with plasma membrane rafts, the transmembrane domain and the immediately adjacent highly cationic cytoplasmic domain are the likely candidates by association with syndecan-1. Of note, the presence of the phosphoinositol 4,5-bisphosphate binding domain in syndecan-4 (absent in syndecan-1) does not inhibit its localization to the raft subdomain. The presence of plasma membrane cholesterol is clearly required for this event as its depletion led to a virtually complete loss of syndecan-4 enrichment in Triton X-100-insoluble regions.

Although syndecan-4 clustering with its signaling agonist FGF2 induces its redistribution to non-caveolae rafts, the functional significance of this event is unclear. One possibility is that this serves to bring syndecan-4 in close contact with FGF receptors. Because FGF receptor-dependent activation of a protein phosphatase is needed for the activation of syndecan-4 signaling (16), syndecan-4 movement into rafts would serve to facilitate this connection. Alternatively, raft localization of syndecan-4 complexes may bring it in close contact with other signaling complexes, thereby promoting cross-talk among various receptor pathways.

In summary, ligand-dependent clustering of syndecan-4 in primary endothelial cells leads to its concentration in non-caveolae rafts. This event may play a role in the regulation of syndecan-4 signaling.

	ACKNOWLEDGEMENTS

We thank K. Williams for Fc receptor Ia cDNA, N. Shworak for syndecan-4 antiserum, Dr. L. Pelkmans for caveolin-1-GFP expression vector, Dr. Justin D. Pearlman for developing of plugins for ImageJ, Dr. Arie Horowitz for helpful discussions of the results, and Alice Givan and Ken Orndorff (Englert Cell Analysis, Norris Cotton Cancer Center, Dartmouth Medical School) for help with life confocal microscopy.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Section of Cardiology, Dartmouth-Hitchcock Medical Center, One Medical Center Dr., Lebanon, NH 03756. Tel.: 603-650-3540; Fax: 603-650-6164; E-mail: michael.simons@dartmouth.edu.

Published, JBC Papers in Press, March 11, 2002, DOI 10.1074/jbc.M200841200

	ABBREVIATIONS

The abbreviations used are: GPI, glycosylphosphatidylinositol; FcR, Fc receptor Ia CD64; FcR-S4, Fc receptor-syndecan-4 chimera; FLAER, Alexa-488-conjugated inactive variant of the protein proaerolysin; HRP, horseradish peroxidase; EGFP, enhanced green fluorescent protein; RFPEC, Rat fad pad endothelial cells; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; FGF2, fibroblast growth factor 2.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. Paris, A. Burlacu, and Y. Durocher Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery J. Biol. Chem., March 21, 2008; 283(12): 7697 - 7704. [Abstract] [Full Text] [PDF]

				A. Ziegler and J. Seelig Binding and Clustering of Glycosaminoglycans: A Common Property of Mono- and Multivalent Cell-Penetrating Compounds Biophys. J., March 15, 2008; 94(6): 2142 - 2149. [Abstract] [Full Text] [PDF]

				C. J. Clarke, V. Ohanian, and J. Ohanian Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2248 - H2256. [Abstract] [Full Text] [PDF]

				M. D. Bobardt, U. Chatterji, S. Selvarajah, B. Van der Schueren, G. David, B. Kahn, and P. A. Gallay Cell-Free Human Immunodeficiency Virus Type 1 Transcytosis through Primary Genital Epithelial Cells J. Virol., January 1, 2007; 81(1): 395 - 405. [Abstract] [Full Text] [PDF]

				K. J. McQuade, D. M. Beauvais, B. J. Burbach, and A. C. Rapraeger Syndecan-1 regulates {alpha}v{beta}5 integrin activity in B82L fibroblasts J. Cell Sci., June 15, 2006; 119(12): 2445 - 2456. [Abstract] [Full Text] [PDF]

				E. Tkachenko, A. Elfenbein, D. Tirziu, and M. Simons Syndecan-4 Clustering Induces Cell Migration in a PDZ-Dependent Manner Circ. Res., June 9, 2006; 98(11): 1398 - 1404. [Abstract] [Full Text] [PDF]

				M. Gopalakrishnan, K. Forsten-Williams, M. A. Nugent, and U. C. Tauber Effects of Receptor Clustering on Ligand Dissociation Kinetics: Theory and Simulations Biophys. J., December 1, 2005; 89(6): 3686 - 3700. [Abstract] [Full Text] [PDF]

				S.-Y. Chang, H.-J. Ko, T.-H. Heo, and C.-Y. Kang Heparan Sulfate Regulates the Antiangiogenic Activity of Endothelial Monocyte-Activating Polypeptide-II at Acidic pH Mol. Pharmacol., May 1, 2005; 67(5): 1534 - 1543. [Abstract] [Full Text] [PDF]

				E. Tkachenko, J. M. Rhodes, and M. Simons Syndecans: New Kids on the Signaling Block Circ. Res., March 18, 2005; 96(5): 488 - 500. [Abstract] [Full Text] [PDF]

				C.-S. Chung, C.-Y. Huang, and W. Chang Vaccinia Virus Penetration Requires Cholesterol and Results in Specific Viral Envelope Proteins Associated with Lipid Rafts J. Virol., February 1, 2005; 79(3): 1623 - 1634. [Abstract] [Full Text] [PDF]

				D. M. Beauvais, B. J. Burbach, and A. C. Rapraeger The syndecan-1 ectodomain regulates {alpha}v{beta}3 integrin activity in human mammary carcinoma cells J. Cell Biol., October 11, 2004; 167(1): 171 - 181. [Abstract] [Full Text] [PDF]

				R. V. Stan, E. Tkachenko, and I. R. Niesman PV1 Is a Key Structural Component for the Formation of the Stomatal and Fenestral Diaphragms Mol. Biol. Cell, August 1, 2004; 15(8): 3615 - 3630. [Abstract] [Full Text] [PDF]

				E. Tkachenko, E. Lutgens, R.-V. Stan, and M. Simons Fibroblast growth factor 2 endocytosis in endothelial cells proceed via syndecan-4-dependent activation of Rac1 and a Cdc42-dependent macropinocytic pathway J. Cell Sci., July 1, 2004; 117(15): 3189 - 3199. [Abstract] [Full Text] [PDF]

				M. D. Bobardt, P. Salmon, L. Wang, J. D. Esko, D. Gabuzda, M. Fiala, D. Trono, B. Van der Schueren, G. David, and P. A. Gallay Contribution of Proteoglycans to Human Immunodeficiency Virus Type 1 Brain Invasion J. Virol., June 15, 2004; 78(12): 6567 - 6584. [Abstract] [Full Text] [PDF]

				C. C. Chua, N. Rahimi, K. Forsten-Williams, and M. A. Nugent Heparan Sulfate Proteoglycans Function as Receptors for Fibroblast Growth Factor-2 Activation of Extracellular Signal-Regulated Kinases 1 and 2 Circ. Res., February 20, 2004; 94(3): 316 - 323. [Abstract] [Full Text] [PDF]

				K. J. McQuade and A. C. Rapraeger Syndecan-1 Transmembrane and Extracellular Domains Have Unique and Distinct Roles in Cell Spreading J. Biol. Chem., November 21, 2003; 278(47): 46607 - 46615. [Abstract] [Full Text] [PDF]

				E. G. Argyris, E. Acheampong, G. Nunnari, M. Mukhtar, K. J. Williams, and R. J. Pomerantz Human Immunodeficiency Virus Type 1 Enters Primary Human Brain Microvascular Endothelial Cells by a Mechanism Involving Cell Surface Proteoglycans Independent of Lipid Rafts J. Virol., November 15, 2003; 77(22): 12140 - 12151. [Abstract] [Full Text] [PDF]

				O. Ben-Zaken, S. Tzaban, Y. Tal, L. Horonchik, J. D. Esko, I. Vlodavsky, and A. Taraboulos Cellular Heparan Sulfate Participates in the Metabolism of Prions J. Biol. Chem., October 10, 2003; 278(41): 40041 - 40049. [Abstract] [Full Text] [PDF]

				K. Mani, F. Cheng, B. Havsmark, M. Jonsson, M. Belting, and L.-A. Fransson Prion, Amyloid {beta}-derived Cu(II) Ions, or Free Zn(II) Ions Support S-Nitroso-dependent Autocleavage of Glypican-1 Heparan Sulfate J. Biol. Chem., October 3, 2003; 278(40): 38956 - 38965. [Abstract] [Full Text] [PDF]

				S. A. Wickstrom, K. Alitalo, and J. Keski-Oja Endostatin Associates with Lipid Rafts and Induces Reorganization of the Actin Cytoskeleton via Down-regulation of RhoA Activity J. Biol. Chem., September 26, 2003; 278(39): 37895 - 37901. [Abstract] [Full Text] [PDF]

H. Slimani, N. Charnaux, E. Mbemba, L. Saffar, R. Vassy, C. Vita, and L. Gattegno Binding of the CC-chemokine RANTES to syndecan-1 and syndecan-4 expressed on HeLa cells Glycobiology, September 1, 2003; 13(9): 623 - 634. [Abstract] [Full Text] [PDF]