(Received for publication, February 13, 1995; and in revised form, July 21, 1995)
From the
We expressed domains of the core protein of the transmembrane heparan sulfate proteoglycan N-syndecan (syndecan-3) either individually or as maltose-binding protein fusion proteins. Biochemical characterization of the purified proteins revealed that some of them were capable of self-association and formed stable, noncovalent multimeric complexes. The formation of N-syndecan core protein complexes was also demonstrated in mammalian cells by in situ cross-linking. Identification of structural motifs in the core protein of N-syndecan responsible for the formation of these complexes was accomplished by analyzing a series of constructs comprising different regions of the protein as well as site-directed mutants. Self-association was assayed by SDS-polyacrylamide gel electrophoresis, glutaraldehyde cross-linking, and size-exclusion high pressure liquid chromatography. Our results indicated that (i) the transmembrane domain of the N-syndecan core protein was required but not sufficient for the formation of stable complexes; (ii) the minimal amino acid sequence that conferred the ability of the N-syndecan core protein to form multimeric complexes included the last four amino acids (ERKE) of the extracellular domain plus the transmembrane domain; (iii) point mutations that changed the basic residues in this sequence to alanine residues either partially or completely abolished the ability of the N-syndecan core protein to form complexes; and (iv) replacement of conserved glycine residues in the transmembrane domain with leucines abolished complex formation. This property is similar to the oligomerization activity of other transmembrane receptors and suggests that regulated self-association may be important for the biological activity of transmembrane proteoglycans.
The syndecans are a gene family of transmembrane cell-surface
heparan sulfate proteoglycans (HSPGs) ()that are expressed
in highly regulated cell type- and development-specific
patterns(1, 2) . Four syndecan core proteins,
designated syndecan-1-4 (also called syndecan, fibroglycan, N-syndecan, and amphiglycan or ryudocan, respectively), have
been identified by molecular cloning from mammalian cells (reviewed in (3) ). The syndecans have structurally distinct extracellular
domains, but highly conserved transmembrane and cytoplasmic domains.
Although the functions of the syndecan family of HSPGs are not known in
detail, they are believed to play important roles in morphogenesis and
differentiation by binding to a variety of extracellular ligands,
including matrix adhesive proteins such as fibronectin and collagens,
and certain polypeptide growth factors (e.g. basic fibroblast
growth
factor)(4, 5, 6, 7, 8, 9, 10, 11, 12) .
The highly conserved nature of the primary structures of the short
cytoplasmic domains of the syndecans strongly suggests that they may,
in addition, play a role in transducing stimuli provided by
extracellular ligand binding into cytoplasmic signals. This could occur
either by binding to and organizing cytoskeletal proteins or by
participating in the generation of intracellular second messengers. In
the case of syndecan-1, association of the core protein with actin
filaments has been demonstrated(13, 14) . Syndecan-4,
but not other members of the family, has been reported to be localized
to focal adhesions in cultured fibroblasts(15) . These are
specialized sites of tight binding of the cell membrane to the adhesive
substratum as well as sites of membrane attachment of intracellular
actin filaments. Several regulatory signaling molecules are
concentrated at focal adhesion sites, including some integrin receptors
and a tyrosine kinase called pp125(16) .
A common feature of signaling mechanisms mediated by cell-surface receptor proteins that contain a single transmembrane domain is the noncovalent dimerization or oligomerization of the proteins in response to ligand binding(17, 18) . Where this phenomenon has been studied in detail, oligomerization appears to be an essential part of the receptor activation process. Oligomerization has been demonstrated both for receptors with cytoplasmic tyrosine kinase domains (e.g. the epidermal growth factor receptor) and for receptors with short noncatalytic cytoplasmic domains. In the latter case, oligomerization may result in the binding and activation of soluble kinases. One consequence of this mechanism of activation is that these receptors can be activated by antibody-mediated cross-linking(19, 20) , which induces oligomerization in the absence of ligand. In addition, it has been shown that some truncated forms of these receptors oligomerize in the absence of ligand binding and are constitutively activated. This suggests that specific domains of these proteins can mediate oligomerization and that other domains inhibit oligomerization until the receptor is activated by ligand binding, presumably as a result of a change in receptor conformation. It is the transmembrane domains of these receptors that are principally involved in mediating oligomerization.
There is indirect evidence to suggest that syndecan family proteoglycans are activated by ligand-mediated cross-linking. In cultured Schwann cells transfected to express syndecan-1, association of the proteoglycan with actin filaments can be induced by antibody-mediated cross-linking of the proteoglycan core protein(14) . This actin association appears to be mediated by the cytoplasmic domain of the proteoglycan since a mutant form of syndecan-1 lacking most of the cytoplasmic domain fails to associate with actin filaments under these conditions.
N-Syndecan (syndecan-3) is a transmembrane HSPG that is
expressed in a fairly restricted pattern, most prominently in the
developing nervous system(21) . This proteoglycan has been
shown to bind basic fibroblast growth factor with high affinity (K = 0.5 nM) (12) and may function as a ``co-receptor'' for this
or related growth factors during nervous system
development(10) . N-Syndecan also binds with high
affinity to heparin-binding growth-associated molecule (HB-GAM), an
extracellular heparin-binding protein with potent neurite
outgrowth-promoting activity(22) .
In this paper, we demonstrate that recombinant N-syndecan core protein self-associates to form stable, noncovalent multimeric complexes in vitro. This property requires the presence of the transmembrane domain as well as a short extracellular domain sequence that is conserved in syndecan core protein sequences.
Site-directed mutations were generated by PCR amplification. Sense orientation primers containing the desired base changes were synthesized with an in-frame EcoRI recognition site at the 5`-end and used for PCR amplification of wild-type cDNA templates with an antisense primer that contained an in-frame stop codon and a PstI recognition site. The products were subcloned as described above. Introduction of the mutations was confirmed by DNA sequence analysis of the plasmids.
Expression of the
MBP-N-syndecan fusion proteins was induced by the addition of
isopropyl--D-thiogalactopyranoside to the bacterial
cultures (final concentration of 0.3 mM) and incubation at 37
°C for 2 h. The cells were lysed by lysozyme treatment and
sonication, and the fusion proteins were purified by affinity
chromatography on an amylose column as described by the manufacturer
(New England Biolabs Inc.). The fusion proteins were further
characterized by Western blot analysis with either anti-MBP antibodies
(New England Biolabs Inc.) or anti-N-syndecan antibodies.
In some experiments, the fusion proteins were incubated with Factor
Xa, which cleaves a specific peptide recognition site located between
the MBP and core protein segments and results in their separation. The
protein was dialyzed overnight against cleavage buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM CaCl, 1 mM sodium azide). The fusion protein
(10 µg) was incubated with 0.2 µg of Factor Xa for 8 h at room
temperature. The reaction was terminated by the addition of SDS to a
final concentration of 2%.
Immunoblot analysis was carried out as described previously. The preparation and characterization of affinity-purified polyclonal rabbit antibodies against bacterially expressed N-syndecan core protein have been reported previously(21) . We also prepared anti-peptide antibodies directed against the COOH-terminal cytoplasmic domain of N-syndecan. A synthetic peptide corresponding to the terminal peptide KQEEFYA was synthesized and covalently coupled to maleimide-activated keyhole limpet hemocyanin (Pierce). The conjugate was injected into rabbits along with a synthetic adjuvant mixture (RIBI Immunochemicals) as described previously(21) . Anti-core protein antibodies were purified by affinity chromatography on a column containing immobilized recombinant core protein coupled to Sepharose beads as described previously. The antibodies reacted specifically with bacterially expressed N-syndecan core protein (data not shown) and N-syndecan expressed in mammalian cells (see below) on immunoblots.
Figure 1: Self-association of N-syndecan fusion proteins. A fusion protein consisting of MBP (shaded ovals) fused to the COOH-terminal 12-kDa fragment of the extracellular domain (open bars), the transmembrane domain (filled bars), and the cytoplasmic domain (hatched bars) of N-syndecan was expressed in bacteria and purified by amylose affinity chromatography. The position of the Factor Xa cleavage site is indicated, along with the molecular masses of the polypeptides predicted from the cDNA sequence. Left panel, the protein (10 µg) was analyzed by SDS gel electrophoresis after being incubated in buffer without(-) or with (+) Factor Xa. The proteins were visualized by Coomassie Brilliant Blue staining. Migration of molecular mass standards is indicated (in kilodaltons). The bands are labeled based on their apparent molecular masses as follows: 116 kDa, fusion protein dimer; 58 kDa, fusion protein monomer; 42 kDa, MBP monomer; 32 kDa, core protein dimer. Right panel, the MBP-N-syndecan fusion protein (10 µg) was analyzed by SDS gel electrophoresis without(-) and with (+) glutaraldehyde cross-linking as described under ``Materials and Methods.'' The arrow on the right indicates the position of migration of the cross-linked high molecular mass product (XL).
In some experiments, higher order SDS-resistant complexes were observed following SDS gel electrophoresis of the MBP-N-syndecan fusion protein. These migrated at 4 times the predicted monomeric molecular mass and presumably represented stable tetramers (data not shown).
The existence of these complexes in the presence of SDS suggested that they were very stable and that larger complexes may be formed in the absence of denaturing detergent. To detect such complexes, we carried out glutaraldehyde cross-linking in a solution that contained the nonionic detergent Nonidet P-40. As shown in Fig. 1, the protein was cross-linked by glutaraldehyde to high molecular mass complexes that failed to enter the polyacrylamide gel. This suggested that the core proteins were present in large complexes containing at least 8 monomeric units.
Figure 2:
In situ cross-linking of N-syndecan core protein in transfected 293 cells. Left
panel, human 293 cells were transiently transfected with cDNA
coding for rat N-syndecan (N-syn) or a control vector
lacking the cDNA insert. Detergent extracts were subjected to
immunoblot analysis with anti-N-syndecan antibodies (directed
against the cytoplasmic domain). Right panel, N-syndecan-transfected cells were incubated with the
bifunctional cross-linker dimethyl
3,3`-dithiobispropionimidate2HCl as described under
``Materials and Methods.'' Detergent extracts were separated
on an SDS-polyacrylamide gel in the absence(-) or presence
(+) of 5% 2-mercaptoethanol (2-ME). N-Syndecan
molecules were detected by immunoblot analysis. The positions of
migration of the core protein monomeric (M) and dimeric (D) forms are indicated. Numbers and arrowheads indicate the positions of migration of molecular mass standards
(in kilodaltons).
When the N-syndecan-expressing cells were incubated with a bifunctional protein cross-linker, the immunoreactive N-syndecan polypeptides were cross-linked to high molecular mass complexes that barely entered the polyacrylamide gel (Fig. 2). When these cross-linked complexes were analyzed on SDS gels in the presence of 2-mercaptoethanol, which resulted in the separation of the cross-linker functional groups, the high molecular mass complexes were not observed. Under these conditions, the dimer and monomer core protein bands were visible, with the former being more prominent. The prevalence of the dimeric form may be explained by the presence of disulfide bonds within the cross-linker that were resistant to reduction. The presence of SDS-resistant dimers and cross-linked high molecular mass complexes was identical to the behavior of bacterially expressed core proteins described above.
Figure 3: Cross-linking analysis of N-syndecan fusion proteins. The MBP-N-syndecan fusion proteins containing the indicated core protein domains were subjected to SDS gel electrophoresis on 10% (constructs 1-3) or 7.5% (constructs 4-7) polyacrylamide gels. Some samples were subjected to glutaraldehyde cross-linking (+) as described under ``Materials and Methods.'' Control samples(-) were incubated in buffer without glutaraldehyde. The proteins were visualized by Coomassie Brilliant Blue staining (constructs 1-3) or silver staining (constructs 4-7). Numbers correspond to the fusion proteins illustrated schematically. Shaded ovals, MBP; open bars, 12-kDa COOH-terminal fragment of the ectodomain; filled bars, transmembrane domain; hatched bars, cytoplasmic domain. Three of the fusion proteins contained only the last four amino acids of the ectodomain (ERKE) or mutant forms of this tetrapeptide (indicated by underlining) plus the transmembrane domain. Arrows on the left indicate the positions of migration of the molecular mass markers myosin (200 kDa), bovine serum albumin (68 kDa), and ovalbumin (45 kDa).
The extracellular domains of syndecan family core proteins are not highly conserved in amino acid sequence. An exception is a short extracellular domain sequence just preceding the membrane-spanning domain that consists of one or two basic residues flanked by acidic residues. A polypeptide that contained this sequence, corresponding to the last four amino acids of the extracellular domain of N-syndecan (ERKE), in tandem with the transmembrane domain produced cross-linked high molecular mass complexes (Fig. 3, construct 5). These results indicated that the transmembrane domain was required but not sufficient for the formation of stable complexes and that additional sequence from the extracellular domain, together with the transmembrane domain, promoted self-association. Surprisingly, as few as four residues from the extracellular membrane flanking sequence sufficed to confer this property.
To provide additional evidence for the existence of stable
multimeric complexes of the core protein, we analyzed the various
fusion protein constructs described above by analytical gel permeation
chromatography on an SEC4000 column. In these experiments, the column
was eluted with buffer containing the zwitterionic detergent CHAPS
(0.5%, w/v), which produces small micelles in solution (M range of 2500-8600). As shown in Fig. 4, the polypeptides that contained the truncated
extracellular domain, transmembrane domain, or cytoplasmic domain
individually fused to MBP eluted from the column as single major peaks
eluting between 10.2 and 10.8 min. These values corresponded to
apparent molecular masses of 46-60 kDa when the column was
calibrated with globular molecular mass standards. These apparent sizes
were in good agreement with the predicted monomeric molecular masses of
these polypeptides. These data were consistent with the results of
glutaraldehyde cross-linking and suggested that these polypeptides
existed as monomers.
Figure 4: Gel permeation chromatography of N-syndecan fusion proteins. MBP-N-syndecan fusion proteins containing the indicated core protein domains were subjected to analytical gel permeation high pressure liquid chromatography on an SEC4000 column as described under ``Materials and Methods.'' The protein concentrations were 1 mg/ml. The column was eluted with 50 mM sodium phosphate, pH 7.5, 0.1% CHAPS, 0.2 M NaCl at a flow rate of 1 ml/min. Proteins were detected by measuring the absorbance at 280 nm with an on-line monitor. Symbols are as described in the legend to Fig. 3.
When the polypeptide containing the truncated
ectodomain in tandem with the transmembrane domain was analyzed by gel
permeation chromatography, two major peaks were detected (Fig. 4). One of these eluted at 10.3 min and corresponded to
the predicted monomeric size of the fusion protein. The other peak
eluted at 5.6 min (column void volume of 5.0 min), which corresponded
to an M > 1
10
, when the
column was calibrated with globular molecular weight standards. The
trailing shoulder of this high molecular weight peak was broad,
indicating the presence of complexes of intermediate size. Very similar
results were obtained when the polypeptide containing the
extracellular, transmembrane, and cytoplasmic domains in tandem was
analyzed and when the polypeptide containing the ERKE sequence in
tandem with the transmembrane domain was analyzed (Fig. 4). The
formation of high molecular weight complexes was not affected by the
ionic strength of the column buffer, up to 1 M NaCl (data not
shown). These results were consistent with the results of
glutaraldehyde cross-linking and indicated that the transmembrane
domain was required but not sufficient for the formation of multimeric
complexes and that the addition of the four flanking extracellular
amino acids was sufficient to confer this property.
The
cross-linking and gel permeation chromatography data indicated that the
size of the stable complexes was very large. The presence of
intermediate-sized complexes detected by gel permeation chromatography
and the presence of SDS-resistant dimers detected by gel
electrophoresis suggested that these high molecular mass complexes were
in equilibrium with intermediates of smaller size. Consistent with
this, the extent of complex formation as determined by gel permeation
chromatography was dependent on the concentration of the protein. As
shown in Fig. 5, when the concentration of protein injected onto
the column was decreased, the fraction of the total protein that eluted
as complexes was decreased. The relative amounts of the large- and
intermediate-sized complexes were also shifted in favor of the latter.
The ratio of multimer to monomer decreased from 2:1 to 0.75:1 with a
decrease in concentration. A rough estimate of the dissociation
constant for the complexes was determined by nonlinear regression
analysis of the areas under the monomer and complex peaks. The K was calculated to be
5
10
M.
Figure 5: Concentration dependence of N-syndecan self-association. The fusion protein containing the 12-kDa COOH-terminal ectodomain fragment and the transmembrane and cytoplasmic domains (shown in Fig. 1) was subjected to gel permeation chromatography. Before injection into the column, the protein concentration was adjusted as follows: A, 1 mg/ml; B, 0.1 mg/ml; C, 0.01 mg/ml. The ratio of multimer to monomer changed from 2:1 to 0.75:1 with a decrease in concentration. The column was eluted as described in the legend to Fig. 4.
Figure 6:
Transmembrane domain amino acid
substitutions inhibit N-syndecan self-association. Top, the amino acid sequences of the N-syndecan
transmembrane and ectodomain flanking regions are shown, and glycines
and alanine are indicated by closed circles. The vertical
bar indicates the beginning of the transmembrane domain. Two of
the conserved glycine residues were replaced by leucine residues, as
indicated (arrows). Bottom left, the mutant (lane
1) and wild-type (lane 2) proteins containing the
truncated ectodomain and the transmembrane and cytoplasmic domains
fused to MBP (equivalent to the construct shown in Fig. 1) were
expressed and analyzed by immunoblotting with antibodies directed
against the N-syndecan cytoplasmic domain. The positions of
migration of the monomeric and dimeric forms of the polypeptides are
indicated. Bottom right, the wild-type core protein (upper
panel) and the Gly Leu substitution mutant (lower
panel) were subjected to gel permeation chromatography. The ratio
of multimer to monomer peak changed from 2:1 for the wild-type
construct to 0.3:1 for the Gly
Leu
mutant.
The data presented here indicate that the core protein of the
transmembrane HSPG N-syndecan self-associates to form stable
dimers, tetramers, and higher order complexes. The presence of these
complexes was demonstrated using several different methods, including
SDS gel electrophoresis, covalent cross-linking, and gel permeation
chromatography. This property required the presence of the
transmembrane domain of the core protein as well as additional sequence
from the ectodomain. A stretch of sequence as short as four amino acids
of the ectodomain contiguous with the transmembrane domain was
sufficient to confer complex formation. Within this four-amino acid
segment, basic residues that are conserved among syndecan family core
protein sequences appeared to be required. Furthermore, bacterial
constructs containing the entire transmembrane and cytoplasmic domains
of N-syndecan were also capable of forming high molecular mass
complexes. ()
Based on the structural requirements in the core protein necessary for complex formation, the association appeared to be mediated by a combination of noncovalent hydrophobic and nonhydrophobic interactions. The basic unit of these complexes appeared to be dimers. On polyacrylamide gels, SDS-resistant dimers and tetramers, but not trimers or hexamers, were observed for polypeptides that were capable of complex formation. Results of glutaraldehyde cross-linking and gel permeation chromatography indicated that in the absence of SDS (but in the presence of either nonionic or zwitterionic detergent), the complexes consisted of a mixture of units of discrete size. The extent of complex formation was dependent on the protein concentration, suggesting an equilibrium between the monomeric and associated forms. Complex formation was also observed with recombinant core protein expressed in a human cell line. This indicated that the ability to form complexes was not a result of bacterial expression of the proteins. Self-association did not involve interchain disulfide bonds since it was not influenced by the presence of 2-mercaptoethanol or dithiothreitol (data not shown) and was observed for constructs that did not contain cysteine residues. The ability of the core proteins to self-associate was not due to nonspecific aggregation of the hydrophobic domain proteins since constructs that contained this domain without added ectodomain sequence behaved as monomers under all conditions examined. Furthermore, substitution of conserved glycine residues within the transmembrane domain with leucines, which would increase the hydrophobicity of the protein, reduced self-association. This also indicated that specific structural features of the transmembrane domain were important for this property.
A tentative
model for the dimerization of the N-syndecan core protein is
shown in Fig. 7. A comparison of the rat syndecan family core
protein transmembrane domains reveals a regular pattern of small and
bulky side chain residues within the NH-terminal half of
the domain. Moreover, this portion of the transmembrane domain is
predicted to assume an extended conformation based on the high
frequency of glycine residues, which are unfavorable to
-helical
structures and favor extended structures, especially in the
NH
-terminal direction(24) . Contributing to the
maintenance of an extended structure within this region are dipeptide
sequences that are highly favorable for this type of structure. In
contrast, the predicted conformation of the COOH-terminal half of the
transmembrane domain is
-helical. The spacing of the side chains
on neighboring polypeptides could allow for interdigitation of the
alternating small and bulky side chain residues and tight packing
within the membrane. This arrangement would be stabilized by
electrostatic interactions among the charged residues at the
extracellular side of the plasma membrane. A similar pattern of spacing
of small and bulky side chains is also found in other single
transmembrane domain receptors that are known to be activated by
oligomerization (Fig. 7). Also supporting this model is our
finding that replacement of two of the transmembrane glycine residues
with bulky side chain amino acids strongly inhibited self-association.
A similar proposal, but one based on regular packing of small residues
within
-helical transmembrane segments, has been made earlier (17, 18) .
Figure 7:
Model for dimerization of N-syndecan core protein. Top, the amino acid
sequences of the ectodomain tetrapeptide and transmembrane domains of
the four rat syndecans are shown. The vertical bar indicates
the beginning of the transmembrane domain. The ectodomain proximal half
of the transmembrane domain is predicted to maintain an extended
conformation based on the prevalence of Gly residues, which are
unfavorable for helical structures and favor extended structures in the
NH-terminal direction, and the presence of dipeptide
sequences that are highly favorable for extended conformation (VL, VA,
GV)(24) . Within this region, the pattern of small and bulky
side chains residues is conserved. Open circles indicate
positions in which only small side chains residues (Ala or Gly) are
found; filled circles indicate positions in which only bulky
side chain residues (Val, Leu, or Ile) are found. Lower left,
shown is a schematic diagram illustrating a model for dimerization
based on interdigitation of paired small (open ovals) and
bulky (filled ovals) side chain residues on adjacent
polypeptides. The association would be stabilized by electrostatic
interactions between charged residues at the extracellular side of the
plasma membrane. Lower right, this pattern of small and bulky
side chain residues is also found in other single transmembrane domain
proteins that are activated by ligand-induced dimerization (e.g. platelet-derived growth factor receptor (PDGFR-B)),
activated by antibody-induced clustering (
-integrin),
or known to form stable noncovalent dimers (glycophorin). Sequences are
from Refs. 17 and 18. LFA, lymphocyte function-associated
antigen.
A common characteristic of
deglycosylated syndecan family core proteins is that they migrate on
SDS-polyacrylamide gels at positions corresponding to apparent
molecular masses that are significantly higher than the molecular
masses predicted from cDNA sequence analysis(3) . This appears
to be due to a structural feature of the core proteins that causes them
to resist denaturation or to bind SDS poorly. Anomalous migration on
SDS gels is also observed for N-syndecan core protein. The
mature core protein has a predicted molecular mass of 45 kDa based
on the cDNA sequence. The migration on SDS-polyacrylamide gels of the
full-length nonglycosylated core protein is consistent with an apparent
molecular mass of
95 kDa. The anomalous migration of the N-syndecan core protein is due largely to the extracellular
domain, especially the proline- and threonine-rich central spacer
domain. This domain, with a predicted molecular mass of
39 kDa,
migrates on SDS-polyacrylamide gels at an apparent molecular mass of 70
kDa.
Heparitinase- or nitrous acid-digested native or
recombinant N-syndecan that has been glycanated migrates as a
broad band at a molecular mass of 110-120 kDa. This discrepancy
of 15-25 kDa between this form and the nonglycosylated core
protein is apparently due to the presence of carbohydrates that resist
heparitinase or nitrous acid digestion.
In most of the
experiments reported here, we utilized core protein fragments that
lacked the extracellular spacer domain and carbohydrates. These
proteins in their monomeric forms migrated on SDS-polyacrylamide gels
and eluted from gel permeation columns in a manner that was very close
to the behavior predicted from their cDNA sequences. Thus, the shifts
in migration or elution that we report here and attribute to protein
self-association are not simply the result of anomalous behavior of
monomeric polypeptides.
The extent to which the ability to
self-associate is shared by other core proteins of the syndecan family
has not been addressed systematically. The structural motifs we
identified that confer self-association upon the N-syndecan
core protein are conserved among the known members of the syndecan gene
family. Deglycosylated syndecan-2 (fibroglycan), with a predicted M of 23,000, migrates on SDS gels as bands of M
45,000 and 90,000(25) , a finding
that is most easily explained by the formation of SDS-resistant dimers
of the core protein, similar to what we have reported here for N-syndecan. The fibroglycan core protein sequence lacks a
basic residue at position -2 (relative to the membrane-spanning
domain) of the ectodomain. Mutation of the lysine residue at this
position in the N-syndecan core protein abolished complex
formation. We have been unable to observe oligomerization of syndecan-1
core protein under conditions in which they can be observed for N-syndecan (data not shown). The formation of complexes was
seen, however, when a chimera consisting of the syndecan-1
extracellular domain and the N-syndecan transmembrane domain
was analyzed (data not shown).
An important unresolved question
concerns the effect of glycosaminoglycan chains on core protein
self-association. Preliminary results of experiments using purified
native N-syndecan from neonatal rat brain (12) indicated that this form of the proteoglycan cannot be
cross-linked to high molecular mass aggregates. However,
these N-syndecan molecules may contain core proteins truncated
at the COOH-terminal ends as a result of membrane
``shedding.'' This has been demonstrated to occur in cell
culture for all syndecans (1) and appears to result from a
proteolytic cleavage of the protein core in the extracellular domain
near the membrane attachment site. Supporting this idea is the finding
that the purified brain N-syndecan molecules fail to react
with an antibody directed against the cytoplasmic domain.
The functional consequences of this syndecan ``activation'' are unclear, although some data indicate that cytoskeletal coupling may be involved. During the spreading of Schwann cells, stably expressed syndecan-1 on the cell surface transiently co-localizes with nascent actin filaments polymerizing around the cell edges(13) . This apparent association of syndecan-1 with cytoskeletal filaments is lost when spreading is completed. Antibody-mediated cross-linking of syndecan-1 on the surface of spread cells restores co-localization of the proteoglycan with actin filaments(14) . The syndecan-1 microfilament association appears to be functional since antibody-mediated aggregation of syndecan-1 in spreading cells results in a redistribution of the actin filaments. In these experiments, antibodies directed against the proteoglycan core protein may be mimicking the self-association mediated by binding an extracellular ligand. The nature of the activation is not known, but self-association of the core proteins within the membrane could result in the formation of new structures that can regulate cytoplasmic activities such as nucleation of actin polymerization. Consistent with this, we found that actin filament association requires the presence on the core protein of the cytoplasmic domain.