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INTRODUCTION |
Heparan sulfate proteoglycans on the surface of cells regulate
cell behavior by binding via their heparan sulfate chains to numerous
protein ligands (e.g. growth factors, chemokines, and proteases) (1). Heparan sulfate can act in diverse roles as either an
inhibitor or a promoter of protein function or, in some instances, it
may act simply to sequester and protect certain proteins at the cell
surface. This ability to fine tune protein function is dependent on the
structure of the heparan sulfate chain, the binding site for heparan
sulfate on the protein, and the availability of the protein-heparan
sulfate complex to interact with other molecules such as high affinity
receptors for growth factors (2-5). Often these high affinity
receptors must be cross-linked to initiate signaling, and the
concentration of heparan sulfate proteoglycans within specific domains
on the cell surface may aid in this process (6).
Heparan sulfate proteoglycans can exist on the cell surface in discrete
subcellular domains, but our knowledge is limited as to how their
specific localization is controlled. For example, in neurons syndecan-2
localizes specifically to synapses, whereas syndecan-3 is concentrated
in axons (7). In polarized epithelial cells, syndecan-1 localizes to
the basolateral surface and is absent from the apical cell surface (8).
Intracellular sorting mechanisms apparently regulate localization of
syndecans in these cells, because the concentration of syndecan-2 on
dendritic spines at the synapse is dependent on regions within the
cytoplasmic domain (9), whereas deletion of the last 12 amino acids of the syndecan-1 cytoplasmic domain results in the expression of the
mutant proteoglycan at both the apical and basolateral surfaces of
Madin-Darby Canine Kidney (MDCK) cells (10). In addition to
proteoglycan core protein domains, heparan sulfate apparently can
affect intracellular sorting mechanisms, because the amount of
glypican-1 present on the apical surface of polarized epithelial cells
is inversely related to the heparan sulfate content of the proteoglycan
(8). Thus, heparan sulfate on glypican may interfere with its
intracellular sorting to apical compartments, or perhaps heparan
sulfate acts to direct sorting specifically to basolateral compartments.
In contrast to intracellular interactions that regulate proteoglycan
targeting to specific domains, much less is known regarding extracellular events at the cell surface that direct proteoglycan distribution. Studies have shown that cross-linking of syndecan-1 or
syndecan-4 extracellular domains at the cell surface can promote sequestering of these molecules within cholesterol-rich,
detergent-insoluble lipid rafts (11, 12). However, a role for heparan
sulfate in proteoglycan targeting at the cell surface has not been
described, although such activity seems likely based on heparan
sulfate's capacity to bind many proteins resident at the cell surface.
Migration of immune cells involves cell polarization, whereby distinct
subcellular domains are formed. The leading edge of the migrating cell
is termed the lamellipodium, and the trailing edge is called the uropod
(13). Receptors that detect and respond to chemoattractant gradients
are located within the lamellipodia, whereas adhesion molecules such as
ICAMs,1 CD44, and P-selectin
glycoprotein ligand-1 (PSGL-1) are located within the uropod (14). In
addition to normal lymphoid cells, we previously reported that myeloma
cells exhibit polarization and that syndecan-1 localizes specifically
to the uropod of these cells (15). Within the uropod, syndecan-1
promotes homotypic cell adhesion and adhesion to the extracellular
matrix and can also interact with and concentrate heparin-binding
growth factors. It was recently shown that osteoprotegerin (OPG) binds
specifically to the uropods of myeloma cells through interaction with
the heparan sulfate chains of syndecan-1 (16). Following binding, the
OPG is taken up by the cell and degraded, implying a mechanism by which
myeloma cells can enhance bone turnover. Thus, the subcellular localization of syndecan-1 to the uropod has an important and distinct
impact on the behavior of tumor cells and, perhaps, on that of other
cells in the tumor microenvironment.
The present study reveals that syndecan-1 is targeted to the uropod via
its heparan sulfate chains. Surprisingly, neither the cytoplasmic nor
the transmembrane domains of syndecan-1 are required for targeting, but
the extracellular core protein domain is important because other
heparan sulfate-bearing proteoglycans (betaglycan and glypican-1) fail
to target specifically to uropods. These results provide further
evidence for functional specificity among genetically distinct heparan
sulfate proteoglycans and demonstrate that heparan sulfate chains can
participate in regulating the localization of proteoglycans to specific
sites on the cell surface, thereby compartmentalizing their function.
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EXPERIMENTAL PROCEDURES |
Antibodies and Reagents--
The following monoclonal antibodies
were used: FITC-labeled mouse anti-human syndecan-1 (CD138) antibody
(clone BB4, Serotec, Oxford, UK) (17); FITC-labeled rat anti-mouse
syndecan-1 antibody (clone 281.2) (18); biotinylated anti-human ICAM-1
(CD54) (clone HA58, BD Biosciences); rabbit anti-rat glypican-1
polyclonal antibody (kindly provided by Dr. Arthur Lander) (19); and
anti-heparan sulfate antibody (clone 10E4, Seikagaku America, Falmouth,
MA) (20). Triton X-100, cholera toxin beta-FITC, cytochalasin-D, and
heparin isolated from porcine intestinal mucosa were purchased from Sigma.
Cell Lines and Transfections--
ARH-77 cells (American Type
Culture Collection, Manassas, VA) were grown in RPMI 1640 medium
supplemented with 5% fetal calf serum. These cells are
Epstein-Barr virus-positive lymphoblastoid cells established from a
patient with plasma cell leukemia. CAG cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. These cells were
established at the Arkansas Cancer Research Center from
Histopaque-1077-separated bone marrow aspirates taken from a myeloma
patient. Constructs used in transfections were prepared, cloned into
expression vectors, and transfected into cells as described previously
(Fig. 1). Cells expressing the
transfected proteoglycans were enriched and purified either by cell
sorting or panning using the appropriate antibody.

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Fig. 1.
cDNA constructs used in this study.
All of the following syndecan-1 constructs were generated using a
murine syndecan-1 cDNA: Syn-1, wild-type syndecan-1 (29);
Syn-1HS47, syndecan-1 having mutated heparan sulfate
attachment sites at amino acids 37 and 45, leaving only a single
heparan sulfate attachment site present at position 47 (30);
Syn-1TDM, syndecan-1 having all three heparan sulfate
attachment sites mutated (30); Syn-1279, syndecan-1
truncated at amino acid 279 which produces a core protein lacking a
cytoplasmic domain (22); Syn/glyp, a chimeric proteoglycan composed of
the syndecan-1 extracellular domain and the glypican-1 GPI anchor (22);
glypican-1, wild-type rat glypican-1 (27); Glyp/syn, a chimeric
proteoglycan composed of the glypican-1 extracellular domain and the
syndecan-1 transmembrane and cytoplasmic domains (22); and betaglycan,
wild-type rat betaglycan containing the human c-Myc epitope tag (37).
Detection of the core proteins expressed by the various constructs
was with the appropriate core protein-specific antibodies described
under "Experimental Procedures."
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Immunostaining--
Cells (5 × 104) were
harvested from culture and gently fixed in pre-warmed (37 °C)
formaldehyde (final concentration, 3.7%) and fixed for 5 min. Cells
were washed twice by adding 1 ml of PBS and centrifuged at 800 rpm for
5 min, followed by resuspension in 100 µl of either FITC-conjugated
or unconjugated primary antibodies at room temperature for 45 min. When
unconjugated primary antibodies were used following removal of the
primary antibody and washing, FITC-labeled secondary antibodies were
incubated with cells for 30 min at room temperature. Cells were washed
and resuspended in one drop of Vectashield mounting medium (Vector
Laboratories, Burlingame, CA), mounted on glass slides, and analyzed
for proteoglycan distribution to uropods as described previously (15)
by fluorescence and phase contrast microscopy using an Olympus BX60
microscope equipped with a BX-FLA reflected light fluorescence
attachment (Olympus America, Melville, NY) and a Nikon digital camera.
Disruption of Syndecan-1 Targeting to Uropods--
For
disruption of syndecan-1 targeting using heparin, exogenous heparin (10 µg/ml) was added to cells for 30 min at 37 °C followed by fixation
and immunostaining. In some studies following incubation of cells for
30 min in heparin, the heparin was washed out, and the cells were
resuspended in PBS followed by incubation in PBS at 37 °C. Analysis
of syndecan-1 localization was performed at 1, 2, and 4 h after
removal of the heparin. To remove heparan sulfate chains from the cell
surface, cells were sequentially treated twice with heparitinase (1 milliunit/ml; Seikagaku America) at 37 °C for 30 min and analyzed
for syndecan-1 localization.
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RESULTS |
Targeting of Syndecan-1 to Uropods Is Dependent on Heparan Sulfate
Chains--
Polarized myeloma cells exhibit a clearly defined
morphological protrusion known as the uropod (Fig.
2A). Immunofluorescence staining of polarized myeloma cells demonstrates that syndecan-1 concentrates on uropods on the surface of myeloma cells (Fig. 2B). Staining of these cells with antibody to ICAM-1, a well
characterized marker for uropods (21), demonstrates co-localization of
syndecan-1 and ICAM-1 on uropods and confirms that syndecan-1 indeed is
localized predominantly on the uropod (Fig. 2, C and
D). This, together with our previous study, confirms that
under physiological conditions syndecan-1 is a marker for uropods
present on polarized myeloma cells (15). However, following exposure of
the cells to exogenous heparin, a dramatic redistribution of syndecan-1
occurs, resulting in relatively equal distribution of syndecan-1 over
the entire cell surface (Fig. 2E) rather than concentration
predominantly on uropods (note uniform cell surface staining of
syndecan-1 in Fig. 2E compared with intense staining on
uropods in Fig. 2, B and C). Removal of heparan
sulfate chains from the cell surface with heparitinase also promotes
redistribution of syndecan-1 over the entire cell surface (Fig.
2F). This redistribution occurs even though the cells
clearly retain their uropods and polarized morphology. Dual staining
with antibodies to syndecan-1 and ICAM-1 confirms that uropod integrity
is maintained in the presence of heparin and that, although syndecan-1
becomes distributed over the entire cell surface, ICAM-1 is retained
predominantly on uropods (Fig. 2, G and H). The
effect of heparin or heparitinase on syndecan-1 distribution is not
limited to a few cells; rather, almost all of the cells treated exhibit
a loss of syndecan-1 concentration within the uropod (Table
I). To confirm that heparan sulfate is
required for syndecan-1 targeting to the uropod, ARH-77 cells were
employed. These cells are often polarized, although we have previously
established that the uropods on ARH-77 cells are not as prominent as
those of CAG cells (15). ARH-77 cells lack syndecan-1 expression, but,
following transfection with a cDNA coding for syndecan-1, the
proteoglycan localizes in the uropod (Fig. 2I). In contrast,
when ARH-77 cells are transfected with a cDNA coding for a mutated
form of the syndecan-1 core protein lacking all three heparan sulfate
attachment sites (Syn-1TDM; triple deletion mutant) (22),
the core protein does not concentrate in uropods but is distributed
over the entire cell surface (Fig. 2J). Together, these
findings demonstrate that heparan sulfate chains are required for
targeting of syndecan-1 to uropods.

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Fig. 2.
Targeting of syndecan-1 to the uropod
requires heparan sulfate chains. Phase microscopy (A)
and immunofluorescent staining for syndecan-1 (B) show that
syndecan-1 is concentrated in the uropod (arrows) with
little staining over the remaining cell surface. Dual staining for
syndecan-1 (C) and ICAM-1 (D), a marker for
uropods, confirms that syndecan-1 localization is predominantly within
uropods. Dual staining was performed using a FITC-labeled antibody to
syndecan-1 and a biotinylated antibody to ICAM-1 followed by incubation
of cells with avidin-Texas Red. Syndecan-1 redistributes over the
entire cell surface following the addition of 10 µg/ml heparin to the
culture media for 30 min (E) or when cells are treated with
heparitinase, an enzyme that strips heparan sulfate chains from the
syndecan-1 core protein (F). Note that, although the
syndecan-1 redistributes, the cells remain polarized with distinct
uropods (arrows). Dual staining of syndecan-1 (G)
and ICAM-1 (H) on cells treated with heparin confirms that
syndecan-1 localizes over the entire cell surface, whereas ICAM-1
remains concentrated on uropods. In ARH-77 cells transfected with
murine syndecan-1, the proteoglycan localizes to uropods
(I). However, when ARH-77 cells are transfected with a form
of the syndecan-1 core protein unable to bear heparan sulfate chains
(SynTDM), the core protein localizes over the entire cell
surface rather than concentrating solely on uropods
(J).
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Table I
Syndecan-1 redistributes following the addition of exogenous heparin or
treatment of cells with heparitinase
Cells were treated with heparin or heparitinase and immunostained for
syndecan-1 expression using antibody B-B4 as described under
"Experimental Procedures." Cells were examined for the pattern of
syndecan-1 expression on the cell surface, and the percentage of cells
exhibiting uropod, partial uropod, or distribution over the entire cell
surface was determined. These patterns of expression were defined as
follows: uropod, heavy concentration of staining on uropods and either
no staining or very weak staining over the remaining cell surface;
partial uropod, focal concentration of staining in uropods but also
weaker staining over the entire cell surface; entire cell surface,
equal staining of syndecan-1 over the entire cell surface with no
concentration in uropods. For each assessment, over 100 cells were
examined and counted.
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To determine whether the effects of heparin on the distribution of
syndecan-1 at the cell surface are reversible, cells were incubated
with heparin for 30 min, washed three times in PBS to remove exogenous
heparin, resuspended in PBS at 37 °C, and analyzed for syndecan-1
distribution. Two hours after the removal of heparin, an accumulation
of syndecan-1 is detected within the uropod and, by 4 h after
removal of heparin, the majority of syndecan-1 is detected solely in
the uropod (Fig. 3,
A-C). The finding that syndecan-1 relocalizes to
the uropod when cells are suspended in PBS in the absence of serum
suggests that targeting to the uropod is not mediated by serum proteins
that cross-link syndecan-1 to each other or to other cell surface
molecules. It is possible that some or all of the syndecan-1 that
concentrates in the uropod following removal of exogenous heparin is
actually newly synthesized rather than redistributed syndecan-1
(i.e. the syndecan-1 that exits the uropod in the presence
of exogenous heparin could be shed or recycled, and the syndecan-1 that
accumulates in the uropod over 4 h could be newly synthesized).
Thus, to further examine syndecan-1 redistribution to uropods following
incubation of cells with exogenous heparin, FITC-labeled antibody to
syndecan-1 was added to cells as a tracer of the cell surface molecule.
As expected, immediately after labeling with antibody, the syndecan-1
is seen over the entire cell surface (Fig. 3D). However,
after only 30 min almost all of the labeled syndecan-1 is detected
within uropods (Fig. 3E). Interestingly, the presence of
antibody bound to syndecan-1 appears to accelerate the targeting
process as compared with relocalization to uropods in the absence of
antibody (compare Fig. 3, E and B). A similar
acceleration of targeting to the uropod is seen with CD43 when it is
engaged by antibody (23). Together, the results support the conclusion
that syndecan-1, once on the cell surface, is actively targeted to the
uropod and that the heparin-mediated disruption of syndecan-1
localization to uropods is dynamic and reversible.

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Fig. 3.
Redistribution of syndecan-1 by addition of
exogenous heparin is reversible and dynamic. Heparin was added to
CAG cells for 30 min and washed out, and the cells were fixed and
stained for syndecan-1 immediately (A) or placed back in the
incubator at 37 °C for 2 (B) or 4 h (C)
and then fixed and stained for syndecan-1. In a separate experiment,
following treatment of cells with heparin, cells were washed and
stained for syndecan-1 at 37 °C and observed either immediately
(D) or 30 min after staining with antibody (E).
In the presence of cytochalasin D (10 µM for 60 min at
37 °C), syndecan-1 does not accumulate within the uropod at 4 h
(F) after removal of heparin. Staining of uropods with
cholera toxin beta-FITC demonstrates that the addition of heparin to
CAG cells does not disrupt lipid rafts within the uropods
(G).
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To examine how the actin cytoskeleton effects syndecan-1 localization
on these cells, we employed cytochalasin D, which blocks actin
polymerization. When cells were exposed to cytochalasin D after removal
of heparin, the syndecan-1 remains distributed over the entire cell
surface even 4 h after removal of heparin (Fig. 3F).
Cytochalasin D is known to disrupt the polarized morphology of
lymphocytes, causing cells to round up and abolish uropods. Thus, this
result indicates that, in the absence of uropods, syndecan-1 fails to
concentrate within any specific region of the cell surface.
Recent studies show that lipid rafts comprise subdomains within uropods
of T lymphocytes (24). To determine whether the addition of heparin
disrupts lipid rafts within the uropod, cells were stained with the
FITC-labeled cholera toxin beta. Staining of uropods with cholera toxin
remained prominent in the presence of heparin, indicating that lipid
rafts remained unperturbed, even though the syndecan-1 escapes the
uropod (Fig. 3G). Thus, heparin apparently does not alter
uropod morphology or disrupt lipid raft organization within the uropod.
Specificity of Syndecan-1 Targeting to Uropods via Heparan
Sulfate--
To determine whether other heparan sulfate proteoglycans
would target specifically to the uropod, ARH-77 cells transfected with
a cDNA for either glypican-1 or betaglycan were examined. Neither
of these proteoglycans share significant homology to the syndecan-1
core protein, yet both bear heparan sulfate chains (25, 26).
Surprisingly, neither of these heparan sulfate-bearing proteoglycans
localize specifically to uropods (Fig. 4,
A and C), rather, they are detected over the
entire cell surface, even on those cells with clearly distinguishable
uropods (arrows). In fact, some of the cells appear to have
proteoglycan concentrated in areas distinct from uropods. Staining with
antibody specific for heparan sulfate chains confirms that these
proteoglycans bear heparan sulfate and that this heparan sulfate is not
concentrated within uropods (Fig. 4, B and D).
Moreover, the lack of detection of any focal concentration of heparan
sulfate in the uropods and the lack of intense staining over the entire
cell surface (Fig. 4, B and D) indicate that the
heparan sulfate-dependent targeting mechanism to the uropod
has not been saturated.

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Fig. 4.
Glypican-1 and betaglycan do not target to
uropods. ARH-77 cells expressing either glypican-1 (A
and B) or betaglycan (C and D) were
immunostained to determine the location of the proteoglycan core
proteins (A and C) or the cell surface heparan
sulfate (B and D). Neither proteoglycan core
protein concentrates in the uropod (arrows), and heparan
sulfate is localized over the entire cell surface. Syndecan-1 bearing a
single heparan sulfate chain at amino acid position 47 (Syn-1HS47) targets predominantly to the uropod
(E).
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One possible explanation for the difference between syndecan-1 and
these other proteoglycans in their localization could be the number of
heparan sulfate chains present on the core protein. Glypican-1 has
three closely grouped heparan sulfate attachment sites in a region of
the core protein close to the cell membrane (27). It is not known how
many heparan sulfate chains are actually present on each glypican-1
molecule. Betaglycan bears only one heparan sulfate chain (28). In
contrast, syndecan-1 has three sites for heparan sulfate attachment and
is known to have multiple heparan sulfate chains on each core protein
(29). To determine whether localization of syndecan-1 to the uropod
requires multiple heparan sulfate chains, we utilized a mutated form of
syndecan-1 having two of its three heparan sulfate attachment sites
mutated. This mutated syndecan-1 is composed of a core protein having a single heparan sulfate chain attachment site at amino acid 47 (22, 30).
Although it is not as strongly associated with the uropod as wild-type
syndecan-1, most of the proteoglycan does appear to concentrate in
uropods (Fig. 4E). This suggests that the failure of
glypican-1 and betaglycan to localize specifically to uropods is not
due to the fact that they may bear only one heparan sulfate chain.
The Cytoplasmic and Transmembrane Domains of Syndecan-1 Are Not
Required for Targeting of the Proteoglycan to Uropods--
The
syndecan-1 core protein is composed of a cytoplasmic, transmembrane,
and heparan sulfate-bearing extracellular domain. To determine whether
the cytoplasmic and/or transmembrane domains are required for targeting
of syndecan-1 to the uropod, cells were transfected with either a
mutated form of syndecan-1 lacking the coding region for the
cytoplasmic domain (Syn279, see Fig. 1) or with a chimeric
proteoglycan composed of the syndecan-1 extracellular domain linked to
the GPI anchor of glypican-1 (Syn/glyp). Both of these proteoglycans
are known to bear heparan sulfate chains (22). When expressed in either
ARH-77 cells or CAG cells, the Syn279 and Syn/glyp
proteoglycans are found concentrated in uropods (Fig.
5). Moreover, a chimera composed of the
glypican-1 extracellular domain linked to the transmembrane and
cytoplasmic domains of syndecan-1 (Glyp/syn) fails to concentrate in
uropods. Staining with antibody to heparan sulfate confirms that
heparan sulfate chains are present on the Glyp/syn chimeric
proteoglycan (Fig. 5G). Thus, the cytoplasmic and
transmembrane domains of syndecan-1 are not required for targeting
syndecan-1 to the uropod, and the addition of these domains to
glypican-1 does not promote its concentration within uropods.

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Fig. 5.
The transmembrane and cytoplasmic domains of
syndecan-1 are not required for targeting to the uropod. In cells
that express syndecan-1 lacking its cytoplasmic domain, the syndecan-1
targets the uropod, ARH-77 Syn-1279 (A) and CAG
Syn-1279 (B). The same result is seen for cells
that express chimeric proteoglycans containing the syndecan-1
extracellular domain with a GPI anchor, ARH-77Syn/glyp
(C) and CAGSyn/glyp (D). In contrast,
in cells that express a chimera composed of the glypican-1
extracellular domain and the syndecan-1 cytoplasmic and transmembrane
domains, the proteoglycans do not localize specifically to uropods, but
are distributed over the entire cell surface,
ARH-77Glyp/syn (E) or CAGGlyp/syn
(F). Staining of ARH-77 Glyp/syn-bearing cells with antibody
10E4 confirms that this chimeric proteoglycan bears heparan sulfate
chains (G).
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Syndecan-1 within Uropods Resists Detergent Extraction--
When
cells are extracted with Triton X-100 at 37 °C, syndecan-1 is
retained within the uropod (Fig. 6,
A and B). We have previously demonstrated that
cooling cells on ice for 30 min results in the loss of syndecan-1 from
the uropod, with subsequent redistribution of the proteoglycan over the
entire cell surface (15). Upon brief cooling on ice for 5 min, some of
the syndecan-1 remains in the uropod, whereas some is lost to
surrounding membrane surfaces (Fig. 6C). Extraction of these
cells with Triton X-100 results in specific removal of only the
non-uropod syndecan-1 (Fig. 6D). Thus, at 4 °C,
syndecan-1 looses its association with the uropod and becomes easily
extractable with detergent. Similarly, following the addition of
exogenous heparin and redistribution of syndecan-1, the proteoglycan is
readily extracted with detergent, as is the form of syndecan-1 that
lacks heparan sulfate chains (Fig. 6, E and F).
These results indicate that the loss of syndecan-1 localization to
uropods is associated with a change in the way that syndecan-1 is
anchored to the cell.

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Fig. 6.
Syndecan-1 in the uropod resists detergent
extraction. Syndecan-1 localizes to uropods when CAG cells are
maintained at 37 °C (A) and is retained following
extraction with Triton X-100 (B). When placed in buffer at
4 °C for 5 min the syndecan-1 begins to redistribute over the entire
cell surface, although uropod staining is still strong (C).
Extraction of these chilled cells with Triton X-100 removes the
syndecan-1 that has redistributed, whereas the syndecan-1 within the
uropod resists extraction (D). Following exposure of cells
to heparin, detergent completely removes syndecan-1 (E).
Similarly, detergent removes the syndecan-1 core protein that lacks
heparan sulfate chains (Syn-1TDM) (F).
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DISCUSSION |
Targeting of Syndecan-1 to Uropods Requires Heparan
Sulfate--
Localization of syndecan-1 to the uropods of B lymphoid
cells is functionally important, because it promotes both tight cell adhesion and concentration of growth factors (15). In the present study
we describe the surprising finding that this localization requires the
heparan sulfate chains of syndecan-1. Either the addition of exogenous
heparin or the removal of heparan sulfate from the cell surface results
in the loss of specific localization of syndecan-1 to the uropod and
the rapid redistribution of the proteoglycan over the entire cell
surface. In addition, when a mutated form of syndecan-1 lacking heparan
sulfate chains is expressed on cells, it fails to target specifically
to uropods. Although little is known regarding mechanisms that direct
targeting of heparan sulfate proteoglycans to discrete subcellular
domains, our findings clearly establish that heparan sulfate can play a determining role in this process.
Intracellular sorting of proteoglycans to distinct cell surface
compartments can be influenced by both core protein structure and
heparan sulfate chains (8, 10). However, intracellular sorting
mechanisms do not explain targeting to, and retention of, syndecan-1
within the uropods because, when dispersed by exogenous heparin, the
syndecan-1 relocalizes to the uropod once heparin is removed. Moreover,
if the syndecan-1 is tagged with antibody after it is dispersed by
heparin, the labeled syndecan-1 rapidly relocalizes to the uropod.
Together, these results indicate that targeting of syndecan-1 to
uropods involves events occurring at the cell surface and is not solely
due to targeted delivery of intracellular syndecan to the uropod
compartment. Furthermore, in parallel to what we have demonstrated here
for B lymphoid cells, Triton X-100 extraction of syndecan-1 from
Chinese hamster ovary cells is also facilitated by removal of heparan
sulfate chains or addition of exogenous heparin (10). Thus, our
discovery that syndecan-1 organization on the cell surface is regulated
by heparan sulfate may apply to non-lymphoid cells as well.
Targeting of Syndecan-1 to the Uropod Requires Determinants within
the Syndecan-1 Core Protein--
Interestingly, glypican-1 and
betaglycan, two heparan sulfate-bearing proteoglycans unrelated to
syndecan-1, fail to localize to uropods. Thus, the presence of heparan
sulfate chains alone is not sufficient for targeting heparan sulfate
proteoglycans to uropods. The finding that syndecan-1 targets to
uropods, and other heparan sulfate-bearing proteoglycans do not,
suggests that the core protein of syndecan-1 participates in
localization of the proteoglycan, perhaps by complexing with other
molecules. This could occur via interactions between the syndecan-1
ectodomain and other cell surface molecules. In support of this is
evidence that both syndecan-1 and syndecan-4 ectodomain core proteins
have domains that can interact with other molecules (22, 31). It is
speculated that these ectodomains interact with ligands on the cell
surface as part of the formation of molecular complexes, thereby
generating signaling events that influence cell adhesion and motility.
If this is the case in uropods, syndecan-1 could play a pivotal role in
the formation of multi-molecular complexes. This could be critical in
triggering specific signaling pathways as a result of
syndecan-1-mediated adhesion that occurs via the uropod interaction
with adjacent cells or the extracellular matrix.
The localization of syndecan-1 within uropods apparently is not
dependent on a direct interaction between syndecan-1 and the cytoskeleton. This is supported by the finding that syndecan-1 lacking
its cytoplasmic and transmembrane domains (Syn/glyp) localizes to the
uropod. Nonetheless, when wild-type syndecan-1 is localized within
uropods, it apparently does associate with the cytoskeleton. This is
indicated by the finding that syndecan-1 within the uropod is not
extracted with detergent at 37 °C. Further analysis is necessary to
determine the functional relevance of syndecan-1 interactions with the
cytoskeleton within uropods and the nature of the associations between
wild-type syndecan-1 and the cytoskeleton (and whether these
associations occur directly or via molecular complexes within the
membrane or intracellular domains).
An alternative explanation for the difference in localization between
syndecan-1 and the other proteoglycans (glypican-1 and betaglycan) is
that these other proteoglycans bear heparan sulfate chains that lack
structural features necessary to promote their localization to uropods.
Heparan sulfates are linear polysaccharides containing highly sulfated
domains. The exact pattern of sulfation within a sulfated domain and
the number and spacing of sulfated domains along a heparan sulfate
chain are highly variable, thereby generating vast structural and
functional heterogeneity among heparan sulfate chains (3, 32). Although
the structure and function of heparan sulfate chains on identical core
proteins can differ between cell types (33, 34), little is known
regarding differences in heparan sulfate among distinct proteoglycans
within the same cell type. A recent study showed only slight structural differences between the heparan sulfates of syndecan-4 and glypican-1 isolated from rat embryo fibroblasts but no difference in the ability
of the heparan sulfates to bind the Hep II domain of fibronectin (35).
Our previous studies have shown that, when expressed in B lymphoid
cells, betaglycan, like syndecan-1, can mediate cell adhesion via its
heparan sulfate chains (36). These findings support the notion that
different proteoglycan core proteins on the same cell type have heparan
sulfate chains that are functionally similar. However, it remains to be
determined if structural differences in heparan sulfate account for
differences between the localization of syndecan-1 and other heparan
sulfate proteoglycans.
In conclusion, the present study establishes that the targeting of
syndecan-1 to uropods requires the presence of heparan sulfate chains
on the syndecan-1 core protein. Targeting of syndecan-1 to uropods is
reversible and dynamic and requires the syndecan-1 ectodomain core
protein but not the syndecan-1 cytoplasmic and transmembrane domains.
The finding that betaglycan and glypican-1 fail to target to uropods
provides further rationale for the existence of multiple genetic forms
of heparan sulfate-bearing proteins and suggests a unique function of
syndecan-1 within this subcellular membrane domain.