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Laboratory for Glycobiology and Developmental Genetics, Center for Human Genetics, University of Leuven; and Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium
1Correspondence: Center for Human Genetics, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: guido.david{at}med.kuleuven.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSTRUCTURE AND EXPRESSION OF...FUNCTIONS OF THE SYNDECANSCONCLUSIONSREFERENCES |
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Key Words: focal adhesions • microfilaments • PDZ domains • kinases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSTRUCTURE AND EXPRESSION OF...FUNCTIONS OF THE SYNDECANSCONCLUSIONSREFERENCES |
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. The extracellular matrix
(ECM)2-adhesion sites-cytoskeleton axis is proposed to play a crucial role.
Cells are anchored to the ECM and surrounding cells via specific
membrane receptors (integrins, cadherins, selectins, CAMs) that are
clustered within adhesion sites (focal adhesion complexes and
intercellular complexes) and physically coupled to the tensed
cytoskeleton. Cell-generated stresses and external mechanical forces
converge on adhesion sites and the transmembrane receptors present in
these sites provide molecular supports for the transfer of mechanical
signals across the cell surface. The effects of mechanical signals
depend on other environmental signals, such as those emanating from
growth factors and other ECM molecules. Remarkably, the sensors
activated by growth factors are spatially incorporated into focal
adhesion complexes. Growth factors, ECM, and mechanical stresses
activate the same intracellular signaling cascades and many of the
molecules in these pathways are physically immobilized on the
cytoskeleton. This may explain how signaling molecules associated with
the cytoskeleton change their function when force is applied to the
cell surface (4)
. No doubt, decoding mechanotransduction
promises to be very complex. Syndecans are transmembrane proteoglycans implicated in several signal transduction cascades that regulate cell proliferation and are also at the cross-section of mechanical signaling, interacting with various effectors of mechanotransduction. Via their extracellular glycosaminoglycan (GAG) chains they bind a multitude of growth factors and ECM molecules. Via their small cytoplasmic domain they interact with the cytoskeleton and potential downstream signal transducers. Therefore, investigators trying to understand the molecular mechanisms of mechanotransduction and perhaps the effect of microgravity will probably deal with the syndecans at some point. In this review, we focus on the properties, binding proteins, and potential functions of the cytoplasmic domain of the syndecans.
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STRUCTURE AND EXPRESSION OF THE SYNDECANS |
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TOPABSTRACTINTRODUCTIONSTRUCTURE AND EXPRESSION OF...FUNCTIONS OF THE SYNDECANSCONCLUSIONSREFERENCES |
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, 6
). In vertebrates,
the syndecan family comprises four members (Fig. 1A
). The ectodomains of these molecules display low sequence
homology, except for the GAG attachment sites (see below). In contrast,
their single membrane-spanning regions and their cytoplasmic domains
are highly similar (Fig. 1B
). The cytoplasmic domain of each
syndecan has two conserved regions, C1 and C2, respectively proximal
and distal to the membrane and common to all syndecans. The C1 and C2
regions are separated by a variable (V) region unique to each syndecan.
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Syndecan expression is highly regulated and distinctive patterns of syndecan expression characterize individual cell types, tissues, and developmental stages. Virtually all cell types express at least one form of syndecan, but most express multiple syndecans. As an approximate rule of thumb, syndecan-1 is the major syndecan in epithelial cells, syndecan-2 predominates in fibroblasts, syndecan-3 abounds in neuronal cells, and syndecan-4 is widely expressed. The most dramatic changes in syndecan expression occur during development and are associated with morphological transitions, cell differentiation, or changes in tissue organization. Syndecan expression is also modified in pathological situations, such as wound healing and neoplastic transformation (5-9) .
Syndecans are substituted with long, unbranched polysaccharides of the
GAG type. The majority of GAG chains added to syndecan core proteins
are heparan sulfate (HS) chains but some of these proteins bear
chondroitin sulfate chains as well. After attachment of a
tetrasaccharide linkage region to particular serines in these proteins,
the biosynthesis of HS chains is achieved by sequential addition of
alternating D-N-acetylglucosamine and
D-glucuronic acid moieties to the non-reducing end of the
growing GAG chain. The number of disaccharides varies from 50 to 150
units. During and after this assembly, individual saccharide units are
subjected to a number of enzymatic modifications (reviewed in ref. 10
).
The variability of these modifications along the length of the polymer
generates the HS fine structure. This fine structure defines the
functional properties of these molecules, since in many cases the
binding of a particular ligand to HS depends on a particular pattern of
HS modification (reviewed in ref. 11
). This patterning is highly
regulated and characterization of the enzymes responsible for this HS
fine structure as well as the elements that encode the patterning is an
intensive area of research. It is clear that a particular syndecan can
bear differentially modified HS chains depending on cellular origin and
developmental stage (see for example ref. 12
). This adds an additional
level of complexity to the structure and versatility of the binding
interactions of these proteoglycans.
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FUNCTIONS OF THE SYNDECANS |
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TOPABSTRACTINTRODUCTIONSTRUCTURE AND EXPRESSION OF...FUNCTIONS OF THE SYNDECANSCONCLUSIONSREFERENCES |
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. As HSPGs, syndecans are thereby implicated in the
control of cell proliferation, differentiation, adhesion, and
migration, as well as in phenomena like blood coagulation, lipid
metabolism, and infection. Nearly all these HS ligands have additional
receptors that exhibit a higher degree of binding specificity than the
HSPGs and that directly mediate ligand-dependent intracellular
activation steps. The genetic evidence that HSPGs are equally important
and that the dysregulation of these molecules leads to pathology is
compelling. Mice homozygous for a mutant heparan sulfate
2-sulfotransferase gene die as neonates with renal agenesis and
skeletal abnormalities (13)
. Drosophila
N-deacetylase-N-sulfotransferase mutants show
wingless- phenotypes (Lin et al.,
personal communication). How the cell-surface HS operates in these
signaling pathways is currently under intense study. This has been
particularly well investigated in the case of the activation of
tyrosine kinase receptors for the fibroblast growth factor (FGF) family
members, mostly bFGF (reviewed in ref. 9
). Cultured cells
fail to respond to this growth factor if they are treated with
heparinases, which remove HS, or incubated with chlorates, which block
sulfation and thus maturation of the chains. Failure of bFGF to bind
and activate the FGF-receptor kinase also occurs in cells that carry
mutations in genes encoding enzymes involved in HS biosynthesis. This
defect can be rescued if an exogenous source of HS is added, either as
soluble molecule or as HS-proteoglycan on adjacent cell surfaces
(14)
. Different models have been proposed to explain how
HSPGs may influence ligand-receptor interaction and signaling
(9
, 15-19
). HSPGs may catalyze the formation
of receptor-ligand complexes through allosteric and bridging
mechanisms, and affect ligand multimerization and stability. They may
influence ligand diffusion and compartmentalization, concentrating
ligands close to the signaling receptors at the cell surface,
restricting ligands to specific spatial plasma-membrane domains (e.g.,
basolateral pole, focal adhesions), mediating the endocytosis of the
ligands, or sequestering the ligands away from the membrane receptor
once shed from the cell surface. Thus, as co-receptors, the syndecans
can be essential regulators of primary receptor activation. A more
in-depth discussion of this aspect of their function is beyond the
scope of this review.
Functions specific to syndecans
As mentioned above, the transmembrane and cytoplasmic regions of
syndecans are highly conserved. This strongly suggests that syndecans
also play a role in transducing stimuli, provided by extracellular
ligand binding, into cytoplasmic signals, or vice versa. Because their
cytoplasmic domain is non-catalytic, this could occur by binding to and
participating in the organization of cytoskeletal proteins or effectors
of intracellular signal transduction cascades. We will review the
evidence accumulated in this regard. Often these studies relate only to
one particular syndecan, but at least in some cases the conclusions may
reasonably be extended to the other members of the family.
Syndecan-1, cell behavior, and microfilament cross-talk
During development, wound repair, or neoplastic transformation,
changes in syndecan-1 expression parallel changes in cell behavior
(7
, 20
). Similarly, transfection-induced
changes in syndecan-1 expression cause cells to alter their shape,
growth, and migration rates as well as their cytoskeletal organization
(reviewed in ref. 9
). More than a decade ago, membrane
proteoglycans were proposed to interact specifically with F-actin via
their cytoplasmic domains and to provide a transmembrane link by which
ECM binding to cell surface HS may control cell shape
(21)
. Later, syndecan-1 was shown to co-localize with
intracellular microfilaments at the basolateral cell surface of mouse
mammary epithelial cells (22)
.
Subsequent work by Carey and his collaborators on clones of transfected
Schwann cells extended this original observation. Clones overexpressing
syndecan-1 exhibit enhanced spreading and altered morphology on various
substrates, including fibronectin and laminin. This spreading is
accompanied by a reorganization of the cytoskeletal structures and the
formation of focal adhesions. Patches of cell surface syndecan-1
co-localize with actin during cell spreading but this co-localization
is lost when spreading is completed and no stable association of
syndecan-1 with focal contacts is observed (23)
. This
transient co-localization may be due to ligand-mediated clustering of
the syndecan, since antibody-induced aggregation of syndecan-1 at the
cell surface promotes the co-localization of this proteoglycan with the
microfilaments and leads to a reorganization of actin filaments
(24)
. Antibody-induced syndecan association with
microfilaments and effect on microfilament reorganization require the
cytoplasmic domain because these properties are lost when syndecan-1
lacks the last 31 carboxy-terminal amino acid residues. Another study
shows that deletion of the last 23, but not of the last 11,
carboxy-terminal amino acid residues of syndecan-1, as well as the
mutation Y to F in the V region of syndecan-1 abolish the
co-localization with microfilaments, demonstrating that microfilament
association is functionally linked to the V region of syndecan-1
(25)
. There is evidence that clustering of syndecan-3 also
mediates the association of this syndecan with the microfilament system
(9)
. It may be noted that the syndecan-1 and syndecan-3
cytoplasmic domains are highly similar (see Fig. 1B
).
Whether the V region is sufficient to affect microfilament
reorganization was not addressed.
Yet, the cytoplasmic domain is not always implicated in the effects of
syndecan-1 on cell behavior. Transfection of syndecan-1 in human Raji
lymphoblastoid cells, HS-negative suspension-growing cells, allows the
binding and spreading of these cells on thrombospondin, fibronectin,
and immobilized antibodies specific for the ectodomain of syndecan-1
core protein. It is surprising to note that syndecan-1 cytoplasmic
deletion mutants (lacking the last 12 or 33 amino acid residues)
maintain the ability to spread. Nevertheless, spreading is inhibited by
cytochalasin D (which blocks actin polymerization) or colchicine (which
blocks microtubule polymerization) (26)
.
Syndecan-1 is also incriminated in the maintenance of differentiated
epithelial morphology, a well-organized actin filament system, and
normal growth of epithelial cells (see for example refs.
27-29
). Re-expression of syndecan-1 in S115 mouse mammary
epithelial cells that exhibit a transformed phenotype reestablishes the
epithelioid morphology and actin organization of these cells and
restricts their tumorigenic growth. However, these effects are not only
observed with full-length syndecan-1, but also with syndecan-1 forms
that lack the cytoplasmic domain or both the cytoplasmic domain and the
transmembrane domain (30)
.
Thus, the syndecan-1 cytoplasmic domain can, directly or indirectly, mediate interaction with microfilaments and reorganize microfilaments on clustering of the core protein. Nevertheless, in other instances, syndecan-1 can affect the microfilament system without its cytoplasmic domain. The molecular mechanisms responsible are not known. The expression of the syndecan-1 ectodomain may affect spreading mediated by other transmembrane proteins with cytoskeletal linkages or interfere with FGF signaling.
Syndecan-4, focal adhesions, and protein kinase C (PKC) activation
Woods et al. have shown that two signals are needed to
achieve the complete adhesion of primary embryonic fibroblasts to
fibronectin (31)
. The first signal occurs through integrin
binding to the RGD sequence of the cell-binding domain of
fibronectin. This promotes attachment and spreading. The second signal
is mediated by the heparin-binding activity of fibronectin and allows
the formation of focal adhesions and stress fibers. This activity is
mainly contained in the PRARI peptide sequence of the carboxy-terminal
heparin-binding domain of fibronectin. The biological response to this
domain seems to be mediated by cell membrane heparan sulfate
proteoglycans because treatment of the cells with heparinase
significantly inhibits stimulation of focal adhesion formation
(32)
. The observation that syndecan-4 is enriched in and
co-distributes with integrins in focal adhesions of many different cell
types adhering to various ECM proteins (33)
led to the
proposal that syndecan-4 mediates focal adhesion and stress-fiber
formation. The response to the second signal can also be obtained by
stimulation of the cells with phorbol esters (34)
,
indicating that one potential downstream effect of HSPGs is the
activation of PKC. On activation and translocation to distinct
intracellular sites, PKC isozymes (35)
participate in many
different agonist-induced signaling cascades and may be important
regulators of cytoskeletal function (36)
. PKC
has been
localized to focal adhesions of normal but not transformed fibroblasts
(37)
and inhibition of PKC correlates with a lack of focal
adhesion formation (34)
. Couchman and collaborators
further investigated a possible link between PKC and syndecan-4. They
showed that syndecan-4, via its V region, can interact with the
catalytic domain of PKC and stimulate its activity. Neither the V
region of syndecan-2 nor that of syndecan-1 is effective
(38)
. PKC activation was directly correlated to the level
of oligomerization of syndecan-4, in particular the V region
(39)
. Dimerization is not sufficient for PKC regulatory
activity, whereas octamers are more active than tetramers. This
indicates that clustering of syndecan-4, in response to extracellular
ligand binding in vivo, may control the signaling event
(39)
. In addition to PKC, the syndecan-4 V region binds
phosphatidylinositol 4,5-bisphosphate (PIP2)
(39-41)
. PIP2 is an important component of
several intracellular signaling pathways (42)
. It
regulates the function of a number of actin binding proteins and is a
substrate for phospholipase C. PIP2 also activates PKC by
binding to its regulatory domain (43)
and stimulates the
translocation of PKC from the soluble to the particulate fraction
(44)
. The syndecan-4 V region further potentiates PKC
activity induced by PIP2, and PIP2 is able to
induce higher-order oligomeric structures of the syndecan-4 V region.
During cell adhesion, clustering of integrins activates
phosphatidylinositol 4-phosphate 5-kinase, resulting in the
accumulation of PIP2. All this suggests that PKC,
PIP2, and the syndecan-4 V region can form a ternary
complex that both localizes PKC to assembling focal adhesions and
potentiates PKC activity (45)
.
On the other hand, in serum-starved primary chicken embryo fibroblasts
cultured on native fibronectin, syndecan-4 localizes poorly to focal
adhesions, but activation of PKC by serum or
12-O-tetradecanoyl phorbol 13-acetate (TPA) induces the
active recruitment of syndecan-4 to focal adhesions (46)
.
The TPA-induced association of syndecan-4 to focal adhesions increases
with time and is more evident with mature focal adhesions than with the
newly forming ones, suggesting that it represents a late event during
adhesion. The association of syndecan-4 with focal adhesions correlates
also with fibronectin deposition. Thus, the syndecan-4 core protein can
be part of focal adhesion complexes but it is not clear if syndecan-4
is required for focal-adhesion formation or recruited to focal
adhesions under certain conditions.
The conserved cytoplasmic region 1 (C1 region) of the syndecans
interacts with members of the src/cortactin signaling pathway and
tubulin
Heparin-binding growth factor-associated molecule
(HB-GAM/pleiotrophin) is a matrix- and cell surface-associated protein
that promotes neurite outgrowth. Syndecan-3 is a cell-surface receptor
for HB-GAM and is implicated in the activity of this molecule on
neurite outgrowth (47-49)
. HB-GAM interacts with the
heparan sulfate chains of syndecan-3 (50)
.
HB-GAM-dependent neurite outgrowth in syndecan-3-transfected cells is
inhibited by the tyrosine kinase inhibitor herbimycin A and by PP1, a
selective inhibitor of the Src family kinases (49)
. When
the cytoplasmic domain of syndecan-3 is immobilized and used for
affinity isolation, a set of proteins including c-src and Fyn kinases,
cortactin, tubulin, and an unidentified 30-kDa protein are eluted from
the column (49)
. All the binding components can be
displaced with a full-length syndecan-3 cytosolic peptide, but also
with a peptide corresponding to the C1 region, suggesting that these
components are binding candidates for all the members of the syndecan
family. However, when the binding components are separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
binding is analyzed by overlaying with a labeled cytosolic peptide of
syndecan-3, only the 30-kDa polypeptide is highlighted. This suggests
that, except for the 30-kDa protein, these components bind indirectly.
Significantly, syndecan-3-transfected cells grown on HB-GAM show
enhanced phosphorylation of c-src and cortactin. Cortactin has been
identified as a microfilament binding protein and as a major substrate
for v-src (51)
. Src-induced tyrosine phosphorylation of
cortactin results in a decreased ability of cortactin to cross-link
actin (52)
. Cortactin redistributes to the cell periphery
with F-actin on src activation (53)
. Src family kinases
have been implicated in many cellular events where syndecans also have
a role, like cell adhesion and spreading, focal adhesion formation,
cell migration, and FGF receptor signaling (54)
. Thus,
interaction with Src family kinases potentially offers a mechanism for
syndecans to regulate these biological events. The fact that tubulin
was detected among the binding proteins of the cytosolic peptide is
also suggestive for a crosstalk between syndecans and microtubules.
The conserved cytoplasmic region 2 (C2 region) of the syndecans
interacts with the PDZ proteins, syntenin and hCASK
In yeast two-hybrid screens, two binding proteins for the C2 (FYA)
region of syndecans have been identified. Both interact with syndecans
via their PDZ domains. PDZ domains are evolutionarily conserved
protein-protein binding modules, originally identified in
membrane-associated guanylate kinase homologs (MAGUKs). They have been
shown to be widespread among signaling and cytoskeletal proteins
(55)
. PDZ domains are globular domains binding to and
discriminating between short carboxy-terminal motifs of three to four
residues. Some PDZ domains appear to bind many targets and, conversely,
some targets bind several different PDZ proteins. PDZ domains have also
been shown to bind to other PDZ domains, and in one case to an internal
(STV) motif (56)
. PDZ domains are involved in the
organization of cytoskeletal and signaling networks, the clustering of
transmembrane receptors, the targeting of particular proteins to
specific subcellular regions, and the promotion of interactions with
cellular substrates (56, and references therein).
The first PDZ-protein identified to bind the syndecan C2 region,
syntenin, is a novel protein. Syntenin is widely expressed and consists
of 298 amino acids and can be divided in at least three subdomains
(57)
. The amino-terminal region (aa 1–109) contains one
potential SH3-binding motif (PXXP), tyrosine residues that on
phosphorylation potentially constitute recognition sequences for SH2
domains, and several serines and threonines. The amino-terminal region
is followed by two PDZ domains in tandem (aa 110–193 and aa 194–274)
that seem both necessary and sufficient for the binding to syndecans.
Recombinant enhanced green fluorescent protein (eGFP)-syntenin fusion
proteins decorate the plasma membrane and co-cluster with overexpressed
syndecans. Cells overexpressing eGFP-syntenin show numerous cell
surface extensions, suggesting effects of syntenin on
cytoskeleton-membrane organization. Syntenin has no obvious catalytic
domain and therefore is unlikely to have a signaling function by itself
but, because of its structure, it could attach syndecans to signaling
components and to the cytoskeleton. The stoichiometry of the
syndecan-syntenin binding remains to be clarified. The requirement of
coupled PDZ domains and the failure of syntenin to bind free peptide
suggests that the syntenin-syndecan interaction may require a prior
dimerization or clustering of the syndecans. In addition, it is not
clear whether syndecans bind both PDZ pockets in syntenin or if one
pocket could remain available for the carboxy-terminal end of another
membrane receptor or cytosolic protein.
A second PDZ protein interacting with the syndecan C2 region is hCASK
(58
, 59
). CASK is a member of the MAGUK
family. It contains an amino-terminal calcium-calmodulin-dependent
protein kinase-like domain, a PDZ domain (responsible for the binding
to syndecans), an SH3 domain, a protein 4.1 binding motif, and a domain
homologous to guanylate kinase. CASK represents the human ortholog of
the C. elegans LIN-2 protein. LIN-2 is required to localize
the LET-23 receptor tyrosine kinase to the basal membrane domain where
it can respond to the LIN-3/EGF-like ligand and induce a Ras/MAP kinase
signaling pathway required for cell fate determination during vulval
cell differentiation (reviewed in ref. 60
). CASK is
expressed in all human tissues tested so far and localizes to basal,
lateral, or basolateral plasma membrane domains in different epithelial
cell types in rat tissue sections. CASK shows overlapping distribution
with syndecan-1 in sections of different mouse epithelial tissues
(58)
and with syndecan-2 in rat brain sections
(59)
. Thus, syndecan-CASK interaction could be implicated
in the basolateral distribution of syndecans in epithelial cells.
Indeed, the C2 and/or V region(s) of syndecan-1 is/are required for the
steady-state basolateral distribution of this proteoglycan in
Madin-Darby canine kidney cells (61)
. In vitro
binding experiments indicate that CASK also interacts with the
actin/spectrin-binding protein 4.1. This raises the possibility that
CASK can bind to other members of this protein family such as ezrin,
radixin, moesin, merlin, or talin, which are implicated in the
organization of the cortical actin cytoskeleton (62)
. By
extension, syndecans could be connected to the cortical actin network
via CASK. In that case, this would represent a path that connects
syndecans and actin that differs from the one described in Carey et al.
(25)
, where the V, and not the C2, region is responsible
for the aggregation of syndecan-1 along stress fibers.
The transmembrane–cytoplasmic domain of syndecans and
internalization of extracellular ligands
Williams and collaborators, studying the role of HSPGs in
lipoprotein catabolism, showed that transfection of Chinese hamster
ovary cells with expression vectors encoding syndecans, increased the
binding and degradation of lipoproteins enriched in lipoprotein lipase,
a heparin-binding protein (63)
. In addition, they provided
strong evidence that, upon clustering, the transmembrane and
cytoplasmic domains of syndecans mediate the internalization of
extracellular bound ligands. In particular, they showed that
IgG-mediated clustering of a chimeric receptor that consists of the
ectodomain of the IgG Fc receptor Ia fused to the transmembrane and
cytoplasmic domains of syndecan-1 initiates receptor internalization
via a non-coated pit pathway (63)
. Ligand internalization
provides additional means for syndecans to regulate signaling events.
Oligomerization, a prerequisite for syndecan signaling?
Deglycanated syndecans migrate aberrantly during electrophoresis
in SDS-polyacrylamide gels. This has been attributed to the formation
of non-covalently linked SDS-resistant dimers and higher-order
oligomers (5)
. Deglycanated syndecan-2, for example,
migrates as bands of 48 and 90 kDa, corresponding to approximately two
and four times the predicted Mr of the protein,
and re-runs as a mixture of 48- and 90-kDa bands after separate elution
and re-electrophoresis of the initial individual bands (our own
unpublished observation). Ligand-induced dimerization or
oligomerization is a key event in signaling via transmembrane
receptors, and the intrinsic propensity of syndecans to self-associate
may be significant in this respect.
Asundi and Carey (64)
have demonstrated that syndecan-3
core protein oligomerization does not require the cytoplasmic domain,
but the transmembrane domain and ectodomain. The oligomerization
appears to be mediated by intermolecular interdigitation of bulky and
small side chains of amino acid residues in the amino-terminal half of
the transmembrane domain, and by electrostatic interactions between
four extracellular juxtamembranar charged amino acid residues.
Preliminary data, indicating that dimerization-deficient forms of
syndecan-3 do not colocalize with actin filaments after
antibody-mediated clustering in transfected Schwann cells
(9)
, may impart physiological significance to these
findings. The extent to which self-association via this mechanism is
shared by all syndecans was not fully addressed, but syndecan-1 core
protein did not oligomerize under similar conditions, whereas
syndecan-2 and -4 lack a presumably crucial basic residue in their
ectodomain.
Yet, glutathion-S-transferase fused to syndecan-2 or
syndecan-4 from which the cytoplasmic domain has been deleted also
migrates with Mr much greater than predicted,
confirming the formation of SDS-resistant oligomers and further
documenting that the cytoplasmic domain is not essential or solely
responsible for self-association (39
, 65
). In
addition, the cytoplasmic domain on its own can also multimerize.
Peptide corresponding to the complete syndecan-2 cytoplasmic domain is
able to dimerize in vitro and this dimerization influences
the extent of the phosphorylation of this peptide by PKC
(65)
. As discussed above, the degree of oligomerization of
syndecan-4 V region correlates with the effect on PKC activity.
Interaction of syndecans with syntenin seems to necessitate
multimerization of syndecans. Thus, syndecan multimerization, which may
be driven by different mechanisms and via different subregions, seems
associated with cytoskeletal interaction and signaling.
Do natural ligands mediate syndecan oligomerization? Adding soluble
lipoprotein lipase and bFGF to cultured fibroblasts induces a
redistribution and alignment of the syndecans with the microfilament
system (66)
, and adding beads coated with bFGF or type I
collagen to syndecan-1-expressing Schwann cells results in the
clustering of syndecan-1 at the cell/bead interface and recruitment of
actin filaments (9)
.
Phosphorylation of the cytoplasmic domain of the syndecans
Phosphorylation events are often crucial in the propagation of
intracellular signals. The cytoplasmic domain of the syndecans contains
three conserved tyrosines, one conserved serine, and various
non-conserved serine/threonines that may serve as phosphorylation sites
(Fig. 1B
). These include consensus sequences for PKA, PKC,
PKG, and calcium-calmodulin-dependent protein kinase II. Among the
conserved tyrosines, two are in a favorable context to be
phosphorylated (67-69)
. One is located in the C1 region
within the sequence DEGSY, the other in the C2 region
within the sequence EFYA. This has led to the speculation
that phosphorylation is a means of regulation. By analogy with other
systems, tyrosine phosphorylation of the syndecan cytoplasmic domains
may provide docking sites for proteins containing SH2 domains and
promote the assembly of signaling complexes at the cell surface.
Although various groups reported that syndecans can be phosphorylated
on their cytoplasmic domain, the physiological significance of the
identified phosphorylations is still not known.
Syndecan-3 tyrosine phosphorylation has been demonstrated in bacteria
expressing Elk kinase (70)
. Syndecan-1 tyrosine
phosphorylation has been reported in vivo after treatment of
cells with pervanadate, a potent inhibitor of tyrosine phosphatases and
activator of cellular tyrosine kinases that are normally retained in
inactive forms by dephosphorylation. It is interesting to note that
pervanadate also induces shedding of the syndecan-1 ectodomain into the
medium, presumably by activation of a membrane protease via
intracellular signaling. Tyrosine phosphorylation-independent shedding
of syndecan-1 can be induced by PKC activation (71)
. Most
cells have only very low steady-state levels of syndecan-tyrosine
phosphorylation but constitutive tyrosine phosphorylation of the
cytoplasmic domain of the syndecans occurs in adherent B82 fibroblasts
(Ott and Rapraeger, personal communication).
In vitro assays with PKCß have shown that, among the 13
serines and threonines available in the four syndecan cytoplasmic
domains, only syndecan-2 S197 (GERKPSSAA) and syndecan-3
S339 (EEPKQASVT) could be phosphorylated by this enzyme
(72)
. In vitro experiments by Oh et al.
(64)
identified S197 and S198 of syndecan-2 as residues
that can be phosphorylated by PKCß
. They also showed that the
extent of phosphorylation varied with the oligomerization state of this
substrate. Syndecan-2 cytoplasmic domain serine phosphorylation has
also been reported in vivo in mouse lung carcinoma cells
(73)
. Horowitz and Simons (74)
observed that
approximately one-third of syndecan-4 S183 is phosphorylated in
growth-arrested NIH 3T3 fibroblasts. This phosphorylation is increased
by phorbol myristate acetate treatment and decreased by bFGF. Based on
the effect of different inhibitors, the authors concluded that
phosphorylation of this serine residue is controlled by a nPKC isozyme
and a bFGF-dependent serine/threonine phosphatase type 1 or 2A. This
serine is part of a conserved seven-residue sequence
(KKDEGSY) in the C1 region and these findings may thus be
relevant to all four members of the syndecan family.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONSTRUCTURE AND EXPRESSION OF...FUNCTIONS OF THE SYNDECANSCONCLUSIONSREFERENCES |
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summarizes the molecular links that are established so far between the
cytoplasmic domains of the syndecans and intracellular kinases,
phospholipids, adaptor proteins, and cytoskeletal proteins.
Nevertheless, the exact role(s) of each of these domains remains far
from obvious. The C1 and C2 regions likely fulfill common roles,
whereas the V region probably serves a unique function. Further
experiments will probably establish additional links between the
cytoplasmic domain of syndecans and intracellular molecules. The
challenge is now in defining the experiments that should establish the
functional relevance of the interaction of syndecans with their
cytoplasmic partners. Concerning a possible sensitivity of syndecan functions to microgravity, the debate is now open. Both the cytoskeleton and intracellular signal transduction, in particular PKC pathways, are modified in microgravity (75-79) . The link between syndecans and these systems has been clearly documented above. Obviously, the role of syndecan-4 in focal adhesion formation and in PKC activation is of special interest in this respect. But are the small changes, in terms of energy, that occur in microgravity likely to have any effect on this syndecan function? The importance of syndecan oligomerization for most syndecan-signaling pathways suggests this step or the tethering of cytoplasmic molecules to the oligomerized cytoplasmic domains could be a privileged target. Syndecan oligomerization is probably dependent on the neutralization by ligand of the highly negative charges, present in HS chains, for which it is difficult to imagine any effect of microgravity, unless the actual signaling form of the syndecans consists of the sole transmembrane and cytoplasmic domains severed from the ectodomain as a result of proteolytic cleavage (shedding). Although receptor processing is part of other signaling systems, for syndecans this remains an unexplored possibility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSTRUCTURE AND EXPRESSION OF...FUNCTIONS OF THE SYNDECANSCONCLUSIONSREFERENCES |
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