Article |
Address correspondence to Arie Horowitz, Angiogenesis Research Center and Section of Cardiology, HB-7504, Dept. of Medicine, Dartmouth Medical School, One Medical Center Dr., Lebanon, NH 03756. Tel.: (603) 650-2635. Fax: (603) 653-0510. E-mail: arie.horowitz{at}dartmouth.edu
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Abstract |
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Key Words: FGF; PDZ; signal transduction; syndecan-4; PKC
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Introduction |
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The potential role of the HS-carrying core of the proteoglycans in mediating the cellular response to growth factors received little attention, however, despite earlier indications of their response to extracellular signals, such as the recruitment of syndecan-4 to focal adhesions (Woods and Couchman, 1994; Baciu and Goetinck, 1995). The possibility of fulfilling specific functional roles seems particularly relevant to the syndecan core proteins, all of which share a distinct and highly conserved cytoplasmic tail. One of the motifs common to all the syndecans is a COOH-terminal postsynaptic density 95, disk large, zona occludens-1 (PDZ)-binding motif now known to bind at least four PDZ domaincontaining partners (Grootjans et al., 1997; Cohen et al., 1998; Hsueh et al., 1998; Ethell et al., 2000; Gao et al., 2000). Similar to other PDZ proteins, these binding partners very likely serve as adaptors between the syndecans and additional members of larger complexes.
Syndecan-4, the most widely spread member of the family, differs in its sequence from the other three syndecans by a unique phosphatidylinositol 4,5-bisphosphate (PIP2)binding seven-residue motif located in the middle of its 28amino acidlong cytoplasmic tail (Lee et al., 1998; Horowitz et al., 1999). Syndecan-4 has been implicated in signal transduction (Volk et al., 1999) and the activation of PKC (Oh et al., 1997b). PIP2 appears to underlie the signaling activity of the cytoplasmic tail of syndecan-4, serving as a binding interface (Horowitz et al., 1999), an essential cofactor for PKC
activation (Oh et al., 1998), and a facilitator of the tail's multimerization (Oh et al., 1997a). These properties are regulated by the phosphorylation of S183 located four residues away from the NH2 terminus of the PIP2-binding motif (Horowitz and Simons, 1998a). Once phosphorylated, the affinity of the cytoplasmic tail for PIP2 and its capacities to oligomerize and activate PKC
in the presence of PIP2 are sharply reduced (Simons and Horowitz, 2001).
Given the HS dependence of the activity of several growth factors and the in vivoobserved increase in syndecan-4 expression during growth factor-regulated healing from injury (Gallo et al., 1994; Nikkari et al., 1994; Li et al., 1997), we asked whether the molecular attributes of syndecan-4 listed above are relevant to growth factor signaling. Using FGF2 as an HS-binding growth factor prototype, we found that disruption of either the PIP2 or PDZ-binding domains of syndecan-4 conferred a dominant negative phenotype in regard to this growth factor but not to serum or epithelial growth factor (EGF)- and PDGF-induced response.
Therefore, these data suggest that syndecan-4 selectively regulates FGF2 signaling in endothelial cells.
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Results |
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To find whether these mutations perturbed FGF2 signaling, we examined several potentially susceptible cell functions. Migration rate in response to FGF2 treatment was measured by a wounding assay of confluent cell monolayers. The migration rates of cells expressing either PIP2- or PDZ- syndecan-4 mutants were similar to each other and threefold lower than the migration rate of vector-transfected RFPECs (Fig. 2 A). At the same time, migration of cells carrying the combined PIP2-/PDZ- mutation was not different from controls. In agreement with our previous observations (Volk et al., 1999), the migration rate of RFPECs overexpressing S4 was even higher than vector-transfected cells (relative gap closure of 0.34 ± 0.09 versus 0.27 ± 0.03, respectively; n = 12, p = 0.023). Cell growth in response to FGF2 over a period of 3 d was measured by proliferation assays with the same cell clones used in the migration assays. Cells expressing either the PIP2- or PDZ- mutants had 35-fold lower proliferation rates in comparison to vector-transfected cells, whereas cells expressing the PIP2-/PDZ- mutation had the same proliferation rate as vector controls, a pattern similar to the results of the migration assays (Fig. 2 B). Finally, tube formation on extracellular matrix basement in response to FGF2 by cell clones expressing the PIP2- or PDZ- syndecan-4 mutations, but not the PIP2-/PDZ- mutation, was also markedly impaired in comparison with the continuous and articulated tube networks formed by vector-transfected cells (Fig. 2 C) or by S4-overexpressing RFPECs (unpublished data).
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PIP2- mutation impairs syndecan-4 targeting to the basolateral region
To elucidate the mechanism of the dominant negative effect of the mutations in the cytoplasmic tail of syndecan-4, we compared the cellular distribution of each mutated syndecan-4 variant to that of the endogenous molecule. Staining of untransfected RFPECs with the antibody to syndecan-4 demonstrated the presence of the proteoglycan along the cell borders and in the perinuclear region (Fig. 3, top). S4 expressed in RFPECs assumed a similar distribution (Fig. 3, bottom). The perinuclear location coincided with the Golgi apparatus as shown by overlap with the staining for the Golgi scaffold protein GM130 (Nakamura et al., 1995).
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PIP2- and PDZ- mutations reduce syndecan-4associated activity of PKC
An established signaling role of syndecan-4 is the modulation of FGF2-stimulated PIP2-dependent PKC activity (Oh et al., 1997b; Horowitz and Simons, 1998a). Since the activity of this PKC isoenzyme has been linked previously to the promotion of cell growth (Kolch et al., 1993, 1996; Cai et al., 1997; Schonwasser et al., 1998; Lallena et al., 1999; Besson and Yong, 2000), migration (Harrington et al., 1997), and tube formation (Wang et al., 2002), we studied the effect of PIP2- and PDZ- mutations on PKC
activity in these cells. To this end, we assayed the activity of PKC
coimmunoprecipitated with HA-tagged syndecan-4 core proteins from the RFPEC clones expressing S4, PIP2-, PDZ-, or PIP2-/PDZ- syndecan-4 constructs, before and after FGF2 stimulation.
FGF2 treatment increased syndecan-4/PIP2-dependent PKC activity eightfold in syndecan-4overexpressing RFPECs relative to untreated cells of the same type (Fig. 6 A). On the other hand, syndecan-4dependent PKC
activity in cells expressing PIP2-, PDZ-, or PIP2-/PDZ- syndecan-4 constructs was not increased by FGF2. No significant FGF2-induced relative increases in syndecan-4associated kinase activities were observed when instead of PIP2 the assays were done in the presence of Ca2+, diacylglycerol, and phosphatidylserine. The absolute level of the Ca2+-dependent activity of PKC
is typically higher by 20% than its activity in the presence of PIP2 and the cytoplasmic tail of syndecan-4 (Horowitz and Simons, 1998a). The fact that the Ca2+-dependent PKC
activities of all of the cell lines tested did not significantly differ from each other (Fig. 6 A), either with or without FGF2 treatment, indicates that similar amounts of PKC
were immunoprecipitated in all cases. Similar to the functional assays described above, the increase in the syndecan-4associated kinase activity was specific to FGF2, since no significant differences were found between the syndecan-4associated kinase activities in serum-treated versus untreated cells. Since the kinase activities were measured ratiometrically for each cell line (FGF2 or serum-treated versus untreated cells), they were not affected by variations between different cell lines in the absolute amounts of syndecan-4associated PKC
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PDZ- syndecan-4 is hyperphosphorylated after FGF2 stimulation
Surprisingly, the FGF2-stimulated PKC activity in cells transfected with the PDZ- mutant was similar to that seen in cells transfected with the PIP2- mutant and much lower than in cells transfected with the native syndecan-4 construct (Fig. 6 A).
Given that the capacity of syndecan-4 to activate PKC is sharply reduced by the phosphorylation of S183 in its cytoplasmic tail (Horowitz and Simons, 1998a), we compared the effect of FGF2 stimulation on the phosphorylation level of the PDZ- mutant to its effect on the S4 variant. FGF2 administration lowered the phosphorylation levels of S4 in a statistically significant manner (Fig. 7) relative to its basal level, in agreement with previously described results (Horowitz and Simons, 1998a), but it did not lower those of the PDZ- mutant. Moreover, the phosphorylation level of the PDZ- mutant after FGF2 treatment was significantly higher than its basal level. This increase may reflect the dependence of the phosphorylation level on a dynamic balance between the opposing actions of a kinase and a phosphatase. The dephosphorylation of the PDZ- mutant would be impaired if the association of syndecan-4 with the putative phosphatase was mediated by a PDZ adaptor protein. Consequently, the activity of the kinase that phosphorylates Ser183, which could conceivably be elevated by FGF2, would be unopposed by the phosphatase, resulting in an effective increase in the phosphorylation level of syndecan-4.
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PIP2- mutation reduces syndecan-4 PIP2-dependent oligomerization and activation of PKC
The capacity of syndecan-4 to activate PKC is dependent on its affinity to PIP2 and on its tendency to oligomerize in the presence of this phosphoinositide (Horowitz and Simons, 1998a). The potent suppression of PKC
activity and of several cell functions by the LQQ mutation implies that the PIP2- mutant has a dominant negative effect when expressed on a background of endogenous syndecan-4. Therefore, we investigated the effect of mixing PIP2--mutated cytoplasmic syndecan-4 tail peptide with the WT tail peptide on the combined affinity of the mixture to PIP2 in vitro. As little as 25% of PIP2- peptide in the total WT/PIP2- mixture increased the apparent KD of the mixture by three orders of magnitude compared with that of the WT peptide alone as measured by surface plasmon resonance (Fig. 8 A).
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Discussion |
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Our working model of this mechanism (Fig. 9) consists of the following steps. (a) FGF2 binding to its high affinity tyrosine kinase receptor induces the activation of a putative serine/threonine protein phosphatase type 1/2A (PP1/2A) (Horowitz and Simons, 1998b), which is associated with the cytoplasmic tail of syndecan-4 through a PDZ adaptor protein. (b) The PP1/2A dephosphorylates Ser183 in the membrane proximal domain of the syndecan-4 cytoplasmic tail, which is normally maintained at a high basal phosphorylation level. Since the HS chains carried by proteoglycans can reach estimated lengths of 80 nm (Kato et al., 1994), the dephosphorylation is likely to occur in a trans rather than a cis mode in regard to the syndecan-4 molecule carrying the FGF2-binding HS chain. Since syndecan-4 is a transmembrane protein, this dephosphorylation may similarly occur in the Golgi-residing syndecan-4. (c) Dephosphorylation of syndecan-4 sharply increases its affinity to PIP2 (Horowitz and Simons, 1998a). In turn, PIP2 binding facilitates the multimerization of syndecan-4. (d) The clustered syndecan-4PIP2 complex activates PKC, which associates with syndecan-4 through PIP2 (Horowitz et al., 1999).
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A recent study on syndecan-4 knockout mice reported that the proliferative response of skin fibroblasts extracted from these mice did not differ from the response of cells from WT mice (Echtermeyer et al., 2001). The difference between this finding and the significantly reduced proliferation rates of cells expressing the PIP2- or PDZ- syndecan-4 mutants that we observed may stem from each of the following reasons. (a) As explained above, the expression of these mutants interferes with the function of endogenous syndecan-4. This active perturbation of a signaling pathway was not present in the mouse syndecan-4-/- cells. (b) Differences between cell types (fibroblasts versus endothelial cells) used in the two studies. (c) Assuming that Echtermeyer et al. (2001) used the same medium as in their migration assays (the medium used in their proliferation assays is not described), the 2% serum present in this medium may have contained other growth factors that compensated for the impaired response to FGF2.
PIP2- mutation: effect on plasma membrane targeting of syndecan-4
One interesting and unexpected finding in this study is the fact that PIP2 binding rather than the interaction with a PDZ protein targets syndecan-4 to the plasma membrane. The data supporting this observation include immunofluorescence staining showing reduced expression of the PIP2- and PIP2-/PDZ- mutants in the basolateral region and virtual absence of syndecan-4 variants containing the PIP2- mutation from the cell surface as demonstrated by FACS. Similarly, deletion of the PDZ motif in the syndecan-2 cytoplasmic tail did not eliminate its incorporation into the plasma membrane (Ethell and Yamaguchi, 1999).
The Golgi apparatus is a known site of PIP2 synthesis (Godi et al., 1999; Jones et al., 2000), and the involvement of PIP2 in protein sorting is suggested by preliminary observations (Morrow, M.W. and P. Weidman, 2000. Mol. Biol. Cell. 11:S280a). However, the actual targeting mechanism is still unknown. The results of the FACS experiments (Fig. 5 A) show that the PIP2- mutation precluded not only the basolateral targeting but also the overall incorporation of syndecan-4 in the plasma membrane.
The dominant negative phenotype exhibited by the PIP2- mutant suggests that it disrupts a syndecan-4dependent protein complex involved in mediating FGF2 signaling. Interestingly, it is the presence of the PIP2- mutant and not a mere absence of syndecan-4 from the cell that impairs cellular function. This conclusion is supported by studies of the PIP2-/PDZ- mutant that does not cause a similar dominant negative phenotype, yet it is similarly absent from the plasma membrane (Fig. 4 C), and by a recently described syndecan-4 knockout mouse (Ishiguro et al., 2000). Instead, we propose that the dominant negative effect of the PIP2- mutation is caused by sequestration to the Golgi apparatus of a syndecan-4binding PDZ protein specifically involved in FGF2 (and not serum, EGF, or PDGF) signaling. Assuming that FGF2 signaling requires the presence of this syndecan-4bound PDZ protein in the basolateral region, the PIP2- mutant may compete with endogenous syndecan-4 for binding to the PDZ protein, thus capturing a large part of this protein population at the Golgi apparatus and interfering with its targeting to the plasma membrane and the formation of a syndecan-4dependent complex.
PDZ binding regulates syndecan-4 phosphorylation in response to FGF2
We have shown that the dominant negative effect of the PDZ- mutation is associated with increased phosphorylation of the PDZ- syndecan-4 mutant after FGF2 treatment. The phosphorylation state of syndecan-4 is controlled on one hand by a novel PKC (nPKC) and on the other by a FGF2-activated PP1/2A (Horowitz and Simons, 1998b). Since the phosphorylation level of the PDZ- mutant was significantly higher than that of S4 after FGF2 treatment, the most likely explanation for this observation is impaired dephosphorylation of the PDZ- mutant, resulting in unopposed activity of the nPKC. This in turn suggests that the PP1/2A involved in syndecan-4 dephosphorylation forms a complex with syndecan-4 through a PDZ adaptor protein. In the absence of the PDZ-binding motif of syndecan-4, and hence PDZ adaptor binding, the phosphatase would no longer dephosphorylate the PDZ- mutant. This explanation applies both to the membrane and the Golgi-associated syndecan-4 pools, since the cytoplasmic tail of the latter would protrude into the cytoplasm and be accessible to the phosphatase and kinase.
The hyperphosphorylated PDZ- mutant has reduced tendencies to oligomerize and bind PIP2, resulting in a diminished capacity to activate PKC (Horowitz and Simons, 1998a). Moreover, its presence in the plasma membrane may interfere with the oligomerization, PIP2 binding, and activation of PKC
by dephosphorylated endogenous syndecan-4. This loss of syndecan-4dependent activation of PKC
in response to FGF2 may then account for the dominant negative effects of the PDZ- mutation.
It should be noted that the lower activity of PKC in the presence of the PDZ- peptide compared with the WT peptide (Fig. 8 C) leaves open the possibility that the induction of a dominant negative phenotype by the PDZ- mutation can be caused by reduction in PKC
activity, similar to the PIP2- cells. Although it is not obvious how deletion of a single COOH-terminal residue in the cytoplasmic tail of syndecan-4 produces this effect, the deletion could conceivably affect the conformation of the PIP2-binding motif and hence reduce its affinity to PIP2.
FGF2 specificity of the syndecan-4 signaling pathway
Another intriguing observation in this study is the apparent FGF2 specificity (versus serum, EGF, and PDGF) of the syndecan-4 signaling pathway. This specificity may stem in part from the choice of functional assays used in this study, since both cell migration (Harrington et al., 1997) and proliferation (Schonwasser et al., 1998; Besson and Yong, 2000) have been linked to the activation of PKC, the kinase regulated by syndecan-4. However, not all FGF2-induced signaling events may necessarily be regulated in this fashion. The fact that, unlike FGF2, serum stimulation of cell growth and proliferation is not affected by the PIP2-and PDZ- syndecan-4 mutants suggests that other growth factors present in serum, such as EGF or PDGF AB, may stimulate cell growth and migration independent of PKC
. The lack of increase in PIP2-dependent PKC
activation after serum stimulation (Fig. 6 A) agrees with this observation.
Though all syndecans and glypicans can carry HS chains and may, therefore, bind FGF2, only syndecan-4 selectively modulates FGF2 signaling (Volk et al., 1999; Simons and Horowitz, 2001). The unique nature of the syndecan-4dependent amplification of FGF2 signaling is related to the presence of its cytoplasmic tail, since the expression of chimeras consisting of either glypican-1 (Volk et al., 1999) or syndecan-1 (unpublished data) ectoplasmic domain fused to the transmembrane and cytoplasmic domains of syndecan-4 mimics the positive effects of intact syndecan-4 overexpression on cell proliferation and migration. These findings, together with the results of the current study, suggest that PIP2 binding and the concomitant ability to activate PKC underlie the unique capacity of syndecan-4 among the cell surface proteoglycans to regulate FGF2 signaling.
In summary, we describe a novel signal transduction pathway that involves selective regulation of endothelial cell migration and proliferation by FGF2 via syndecan-4.
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Materials and methods |
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Alternatively, the HA-tagged syndecan-4 cDNA constructs were inserted between the XhoI and EcoRI restriction sites of a bicistronic mammalian expression plasmid (pIRES2-EGFP; CLONTECH Laboratories) that coexpresses EGFP and the inserted cDNA under a single promoter. RFPECs were stably transfected by LipofectAmine 2000 (Invitrogen) and selected by neomycin resistance as a pooled population. To enrich each pool with cells having high expression levels of the transfected protein, only cells in the top 25% of the population as determined by two to three consecutive sorting rounds (see below) were retained and expanded.
Cell function assays
Cells were incubated as described (Horowitz and Simons, 1998a) in M199 medium (Invitrogen) with the indicated supplements. Cell migration was measured by "wounding" assays (Tang et al., 1997) in which cells were grown to subconfluence in 6-well plates and then starved for 24 h in 0.5% serum. The cell layer was scratched with a pipette tip, producing a gap 2-mm wide. The gap width was measured at marked locations from images taken by inverted microscope (TMS-F; Nikon) immediately after the scratching and again 6 h later at the same locations. Proliferation and tube formation assays were done as described (Volk et al., 1999). All experiments were repeated with two clones of cells stably transfected with each syndecan-4 variant. Neomycin was withdrawn from the culture medium at least 24 h before the cell function assays and before all other experiments with stably transfected RFPECs to prevent artifacts caused by neomycin sequestration of PIP2 (Gabev et al., 1989).
FACS
To measure the amount of exogenous syndecan-4 present on the cell surface, cells were dissociated from plates (nonenzymatic solution; Sigma-Aldrich), labeled with 1 µg/ml antibody to HA (Roche) followed by 10 µg/ml of antirat IgG conjugated to R-PE (Jackson Immunologicals), and sorted automatically (FACScan®; Beckton Dickinson). Detection of the surface expression of both exogenous and endogenous syndecan-4 was done similarly using a 1:50 dilution of antiserum to the ectoplasmic domain of syndecan-4 (Shworak et al., 1994) and 10 µg/ml of antirabbit IgG conjugated to Alexa-594 (Molecular Probes). To detect the overall cellular expression of exogenous syndecan-4, cells were scanned at the EGFP-emitted wavelength (488 nm).
Immunofluorescence
RFPECs were plated in chamber slides (Nunc), fixed with 2% formaldehyde in PBS (137 mM NaCl, 4.3 mM Na2HPO4 · 7H2O, 2.7 mM KCl, 1.4 mM KH2PO4, pH 7.3) for 10 min at room temperature, washed twice with PBS, permeabilized with 0.1% Triton X-100/PBS for 10 min, washed again as above, blocked with 3% BSA (Invitrogen) in PBS for 30 min, and incubated with primary antibodies at the indicated concentrations in 1% BSA in PBS for 3 h. The slides were washed four times as above and incubated for 1 h with 10 µg/ml of the appropriate secondary antibodies conjugated either to Alexa 488 or Alexa 594 (Molecular Probes), washed again as before, and mounted with ProLong medium (Molecular Probes). Slides were imaged by laser-scanning confocal microscopy (Radiance2000; Bio-Rad Laboratories, Inc.).
Immunoprecipitation
Cells were lysed and immunoprecipitated as described (Horowitz and Simons, 1998a, b) using 80 µl anti-HA affinity matrix (Roche) suspension per 750 µl cell lysate. Alternatively, endogenous or HA-tagged syndecan-4 was immunoprecipitated with 10 µl cytoplasmic tail antiserum (Shworak et al., 1994) or with 5 µg HA antibody (3F10; Roche) per 750 µl cell lysate, respectively, and 40 µl suspension of protein G plus/protein A agarose (Oncogene). Where indicated, glycosaminoglycan chains were digested as described (Horowitz and Simons, 1998b).
Surface plasmon resonance
Experiments were performed as described (Horowitz et al., 1999) using 28amino acidlong (Horowitz and Simons, 1998a) syndecan-4 cytoplasmic tail peptides (Genemed Synthesis) as ligands and PIP2 (Sigma-Aldrich) as analyte.
Column chromatography
Synthetic syndecan-4 cytoplasmic tail peptides (1 µM) either alone or mixed with PIP2 (2 µM) in 0.1 M phosphate, pH 7.4, and 20% acetonitrile were injected (22.5 µl) into a 300 x 6 mm silica (5 µm spheres, 60 Å pores) HPLC (Waters, 515 Pump, 2487 Absorbance Detector)-mounted column (YMC). Elution profiles corresponding to light absorbance at 210 nm were recorded digitally (Millennium32; Waters).
Kinase assays
With recombinant PKC.
These were performed either in the presence of PIP2 or Ca2+, phosphatidylserine, and diolein as described (Horowitz and Simons, 1998a) using recombinant PKC (120 ng/ml). Kinase activity was quantified by phosphoimaging (Molecular Dynamics) of gel-resolved PKCßI optimal substrate peptide bands. PKC autoinhibitor peptide and Gö6976 were purchased from Calbiochem.
With immunoprecipitated syndecan-4.
Syndecan-4 complexes immunoprecipitated as described above with 5 µg HA antibody per 750 µl cell lysate and 40 µl protein G plus/protein A agarose suspension were used for in vitro kinase assays. After washing in 25 mM Tris-HCl (pH 7.4) buffer, the beads were sedimented, and the buffer was removed and replaced with 30 ml kinase assay buffer containing either PIP2 or Ca2+, phosphatidylserine, and diolein as described (Horowitz and Simons, 1998a). The assay was stopped by adding 10 µl x4 Laemmli sample buffer (final concentration 2% SDS, 10% glycerol, 0.5% ß-mercaptoethanol, 0.004% bromophenol blue, 50 mM Tris-HCl, pH 6.8) and boiling for 4 min. Kinase activity was quantified as in the recombinant PKC assays.
Measurement of syndecan-4 phosphorylation level
RFPECs in 100-mm plates were grown for 24 h in phosphate-free DME (Invitrogen) supplemented with 0.5% FBS and radiolabeled for 4 h with 0.5 mCi/ml [32P]orthophosphoric acid (New England Nuclear). Cells were washed with tris-buffered saline (137 mM NaCl, 25 mM Tris-HCl, pH 7.4) and scraped off in 0.5 ml lysis buffer (150 mM NaCl, 20 mM NaF, 20 mM Na4P2O7, 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 1 mM PMSF, 1% Triton X-100, 50 mM Hepes, pH 7.4) supplemented with protease inhibitor cocktail (Complete; Boehringer). Cell lysates were precleared by incubation with 1 µg nonimmune rat IgG (Sigma-Aldrich) and 20 µl protein G plus/protein A agarose bead suspension at 4°C for 1 h. After agarose bead sedimentation, the cleared samples were supplemented with 80 µl of anti-HA affinity matrix bead suspension (Roche) or with 10 µl cytoplasmic tail antiserum (Shworak et al., 1994) and 40 µl suspension of protein G plus/protein A agarose (Oncogene) and incubated in rotating tubes over night at 4°C. The beads were sedimented, washed three times in heparinase digestion buffer (50 mM NaCl, 4 mM CaCl2, 20 mM Tris-HCl, pH 7.4), and glycosaminoglycan chains were digested as described (Horowitz and Simons, 1998b). The immunoprecipitated syndecan-4 core protein was dissociated from the beads by a 10-min incubation in 40 µl Laemmli sample buffer at 95°C. Samples were resolved on 12% tris-glycine gels (Bio-Rad Laboratories), and the bands corresponding to the cytoplasmic tail of syndecan-4 were identified by immunoblotting with a peroxidase-conjugated antibody to the HA tag (Roche). The bands were excised, and the 32P level was measured by scintillation counter (Beckman Coulter).
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Footnotes |
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Acknowledgments |
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This study was supported in part by the American Heart Association Scientist Development grant 9730282N (to A. Horowitz) and by National Institutes of Health grants HL62289 and P50 HL63609 (to M. Simons).
Submitted: 28 December 2001
Revised: 20 March 2002
Accepted: 26 March 2002
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