(Received for publication, October 3, 1994; and in revised form, November 28, 1994)
From the e:46, D-60528
Frankfurt, Germany
In the present study we have identified the extracellular matrix protein agrin as a major heparan sulfate proteoglycan (HSPG) in embryonic chick brain. Using monoclonal antibodies and a polyclonal antiserum to the core protein of a previously identified HSPG from embryonic chick brain, our expression screened a random-primed E9 chick brain cDNA library. Twelve cDNAs were isolated that were shown to be identical to the chick extracellular matrix protein agrin. Western blot analysis and immunocytochemistry confirmed that agrin is a HSPG that is identical with the HSPG from embryonic chick brain. A polyclonal antiserum to recombinant agrin protein recognized agrin as a diffuse band of over 400 kDa in extracts from brain and vitreous humor. The agrin immunoreactivity on the blot was shifted to a defined band of approximately 250 kDa after treatment of the samples with heparitinase or nitrous acid, and this banding pattern was indistinguishable from immunoreactivity obtained with antibodies to the brain HSPG. We also show that agrin binds tightly to anion exchange beads, indicating that the molecule is highly negatively charged, which is a hallmark of all proteoglycans. Furthermore, the agrin antiserum recognizes the affinity purified HSPG from chick brain and vitreous humor. Immunocytochemistry demonstrated that agrin is expressed in developing brain, and is especially abundant in developing axonal tracts, in a distribution identical to the staining of the brain HSPG with monoclonal antibodies. We also show that the anti-HSPG antibodies stain the synaptic site of the neuromuscular junction, in agreement with agrin expression. Thus, our studies demonstrate that chick agrin is a HSPG that is prominent in the embryonic chick brain. Since previous studies from our laboratories have shown that this proteoglycan interacts with neural cell adhesion molecule, our studies raise the interesting possibility that neural cell adhesion molecule and agrin are interactive partners that may regulate a variety of cell adhesion processes during neural development, including synaptogenesis.
Proteoglycans are a diverse class of macromolecules whose
defining feature is the addition of one or more glycosaminoglycan
(GAG()) sugar chains. The various classes of proteoglycans
are classified based on the type of GAG chain they
carry(1, 2) , and in many cases the identified
functions of proteoglycans are mediated by their GAG
chains(3, 4, 5) . The functions of
proteoglycans include a role in a number of critical developmental
processes, which include the regulation of cell adhesion and
recognition(6, 7, 8, 9) , the
control of cell growth and differentiation via the binding of growth
factors(10, 11, 12) , and the regulation of
gene transcription(13) .
Recent studies have provided insight into the possible functions of proteoglycans in nervous tissue, and have begun to shed light on the molecular properties of neural proteoglycans. These studies indicate a role for nervous tissue heparan sulfate proteoglycans (HSPGs) in the stimulation of neurite outgrowth during neural development(14, 15, 16) , which is likely mediated by the interaction of HSPGs with various cell adhesion proteins in nervous tissue. In terms of function, the binding of HSPG to cell adhesion molecules is thought to augment the adhesiveness of the cell adhesion proteins, and the role of HSPG in the function of the neural cell adhesion molecule (NCAM) has been well documented(17, 18, 19) . Previous studies have demonstrated that HSPG binding to NCAM is required for NCAM-mediated cell adhesion(17, 18) , and recent studies by Akeson and co-workers (19) in which cell surface HSPG was eliminated by heparitinase digestion confirm these observations. It has also been suggested that heparan sulfate binding to NCAM is required for NCAM-mediated homophilic binding(18, 20) , thus serving as a mechanism for modulating the adhesiveness of NCAM during development. Accordingly, the heparan sulfate binding amino acid sequence in NCAM has been identified(21) , and it has been shown that alteration of the heparan sulfate binding domain of NCAM by site-directed mutagenesis abolishes NCAM's adhesive function, thereby providing strong evidence that HSPG plays a critical role in NCAM function(22) .
We have recently initiated studies aimed at identifying and characterizing HSPGs in chick brain, in order to obtain additional insight into how proteoglycans contribute to the development of the nervous system. In particular, we were interested in identifying the chick brain HSPG that interacts with, and modulates, NCAM function. Our studies have shown that a major chick brain HSPG, which contains a 250-kDa core protein, is expressed during early periods of chick brain development and is capable of interacting with NCAM(23, 24) . This HSPG has been immunopurified using a mAb named 6D2, and the purified HSPG has been shown to promote neural cell attachment when adsorbed onto a nitrocellulose substratum(24) . Thus, these data strongly suggest a role for the 6D2 HSPG in the modulation of neural cell adhesion. In the present study we describe the molecular cloning of the HSPG core protein, using antibody expression screening of an E9 chick brain cDNA library. Interestingly, following the isolation of 12 cDNAs using two different antibody preparations, we found that the putative HSPG cDNAs were identical in sequence to the previously published chick agrin cDNA (25) .
Previous studies have shown that the 220-kDa agrin
protein plays a key role in the aggregation of AChRs during
synaptogenesis in the neuromuscular junction(26) . Recent
studies have focused on the molecular characterization of agrin, and
have demonstrated that agrin is a 220-kDa extracellular matrix protein
comprised of multiple polypeptide
domains(25, 27, 28) . For example, agrin has
been shown to contain multiple protease inhibitor and laminin G
homology domains (28) , raising the possibility that these
domains contribute to its function. Agrin expression during development
has also been shown to be particularly prominent in nervous tissue and
especially brain, although its function in this tissue has yet to be
elucidated(25, 27) . It has been suggested, however,
that brain agrin might have similar and/or additional functions in
nervous tissue(29, 30) . As a means of obtaining
greater insight into the biological actions of agrin, putative
receptors for agrin have been the subject of intense investigation.
Recently, -dystroglycan has been identified as a putative agrin
receptor(31, 32, 33) , although it remains
unclear if agrin-
-dystroglycan interactions in brain are necessary
for agrin function. Agrin-induced aggregation of AChRs in muscle is
also inhibited by heparin and related
polyanions(34, 35, 36) , raising the
possibility that HSPG plays a role in agrin function. Thus, our studies
reported here, describing the identification of an NCAM-binding HSPG (24) as agrin, raise the interesting possibility that agrin
plays an important role in the regulation of cell interactions during
nervous system development.
Expression of
agrin in the vitreous body was examined according to published
protocols(23) , either prior to or following heparitinase
treatment. Agrin can be enriched from brain homogenates by binding to Q
Sepharose, as described by Hascall et al.(42) .
Homogenates from 100 E10 chick brains in CMF (see above) were
centrifuged at 15,000 g for 20 min. Sodium chloride,
Triton X-100, and DNase were added to the supernatant to a final
concentration of 0.3 M, 0.5%, and 5 µg/ml, respectively,
and the solution was incubated with Q-Sepharose (Pharmacia, Piscataway,
NJ; 4 ml/200-ml homogenate) in batch. After shaking for 1 h, the beads
were allowed to settle and the unbound supernatant was decanted. After
two rinses with TBS/Tween, the beads were washed 3 times with 150 ml of
0.5 M NaCl, 4 M urea in TBS/Tween. After a short
rinse with distilled water, the beads were eluted with 4 bed volumes
(20 ml) of 1.5 M NaCl. The eluate was lyophilized, taken up
with water, and dialyzed with CMF. Agrin and vitreous body protein were
visualized by electrophoresis on 3.6-14% polyacrylamide gels,
transfer to nitrocellulose, and immunoblotting using 6D2 mAb.
The expression of agrin in the developing chick visual system and neuromuscular junction was conducted according to published protocols (23) . Briefly, chick heads (E10 or adult) were fixed with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 1 h, followed by cryopreservation of the specimen in 25% sucrose in CMF for 4 h. Cryostat sections through the E10 chick head or adult ocular muscle were incubated with primary antibody (undiluted 6D2 hybridoma supernatant or 1:500 diluted polyclonal anti-agrin fusion protein antiserum) for 1 h, rinsed twice with CMF, and incubated with Cy3-labeled goat anti-mouse or goat anti-rabbit secondary antibodies diluted 1:200 for 1 h. After two additional rinses in CMF the sections were coverslipped and examined under an epifluorescent microscope.
Figure 1: Schematic representation of 6D2 cDNAs isolated following screening of an E9 chick brain cDNA library with the 6D2 and 3A12 mAbs to the HSPG or a polyclonal antiserum to the HSPG core protein. Clones pPG1-10 were isolated following screening with the 6D2 and 3A12 mAbs, and clones pPG12, 15, 20, and 22 were isolated using a polyclonal antiserum to the HSPG core protein. Initial nucleotide sequence was obtained for each clone at the 5` and 3` ends, and is indicated for each clone, and then compared to the published chick agrin sequence. All sequences obtained for the clones were found to be identical to the published chick agrin cDNA sequence. Consensus sequences for 6 GAG attachment sites are indicated on the agrin map, and are localized throughout the agrin protein, including in the functional C-terminal domain.
Although our isolation of multiple overlapping cDNAs that are identical to agrin is indicative of agrin encoding a HSPG, and although Southern blots have revealed only a single gene for agrin in chick(25) , we could not rule out the possibility that major differences in the mRNA coding for agrin leads to the expression of agrin as a HSPG in brain. We therefore carried out Northern analysis of agrin mRNA expression in embryonic chick tissues using one of the isolated proteoglycan cDNAs, pPG5. As shown in Fig. 2A, a 10-kb mRNA was identified that was prominent in brain when compared to heart, and this mRNA exhibited a pronounced down-regulation from E9 to E18 of brain development (Fig. 2A), consistent with expression of the 6D2 HSPG core protein(24) . In addition, we could identify a second, approximately 8-kb mRNA expressed in chick brain glia, which we have shown previously to consist primarily of astrocytes(39) . In previous studies, we have demonstrated that the 6D2 HSPG is expressed by glia in chick brain(24) . These data therefore show that the putative HSPG cDNAs identify a mRNA that parallels the HSPG protein in expression. However, these data also suggest that the HSPG mRNA is larger in molecular size than the previously reported 8-8.5 kb agrin message in chick(25) , although this discrepancy is likely to be attributable to experimental variation between different laboratories, especially in light of a recent study by Ma et al.(44) showing a chick brain agrin mRNA that migrates at approximately 9.5 kb.
Figure 2:
Northern analysis of agrin mRNA
expression. A, analysis of agrin mRNA expression using clone
pPG5. Poly(A) RNA (1 µg) from E9 and E18 chick
brain, E9 heart, and chick brain glia cultured from E10 chick brains
for 1 week, was separated on a 1% agarose gel and blotted with pPG5.
Note that two bands can be discerned with the agrin probe in the glial
mRNA. B, analysis of agrin mRNA expression in E9 and E18 chick
brain using clone pPG12. This blot was originally probed with a mouse
-actin DNA probe (bottom panel), and was then stripped
and probed with the pPG12 cDNA. The analysis with the pPG12 cDNA
represented the third time the blot had been probed with different
cDNAs. The data from this analysis, however, show that the
down-regulation of agrin mRNA with brain development is specific, since
actin mRNA levels show no change.
Because our initial cloning studies using mAbs suggested that agrin encodes a HSPG in brain, and our Northern analyses are consistent with agrin being expressed in brain as a single molecule and hence a proteoglycan, we extended our analyses to confirm that agrin encodes a brain HSPG. Our approach to confirming that agrin encodes a brain HSPG was to carry out a second screening of our random-primed E9 brain cDNA library using a polyclonal antiserum that was prepared to gel purified HSPG core protein. As shown in Fig. 3A, when the 6D2 HSPG is immunopurified using the 6D2 and 3A12 mAbs and analyzed by silver staining, the HSPG and a minor high molecular mass 220-kDa contaminant are isolated. It is important to emphasize that when analyzed on a single percentage gel, rather than a gradient gel, one cannot detect significant amounts of agrin in the absence of treatments to eliminate HS from the HSPG (Fig. 3). Thus, in previous analyses of agrin, which utilized single percentage gels and did not detect intact agrin(27) , one can conclude that this occurred due to the inability of the HSPG to enter the running gel. However, as shown in Fig. 4and Fig. 6, on gradient gels the HSPG can be resolved as a heterogeneous smear with a molecular mass in excess of 400 kDa, in the absence of any treatments to remove HS from the molecule.
Figure 3: Immunopurification of 6D2 HSPG from chick brain. A, silver stain analysis of immunopurified 6D2 HSPG protein electrophoresed on a 6% SDS-PAGE gel. Prior to treatment (U), the HSPG migrates at the stacking and running gel interface (arrowhead), and small amounts of a 220-kDa protein contaminant (small asterisk) are detectable. Following nitrous acid treatment (T), the HSPG core protein is observed (large asterisk). B, immunoblot analysis of immunopurified HSPG protein, prior to or following nitrous acid treatment. It can be seen that the 220-kDa contaminant is also detected by the 6D2 mAb, and that the HSPG core protein is the major protein component. The intact HSPG in the absence of treatment is not readily discernible at the running/stacking gel interface (arrowhead), and results from inefficient transfer from the gel in this region.
Figure 4: Specificity of a polyclonal antiserum to the 6D2 HSPG core protein. A rabbit antiserum generated against the immunopurified 6D2 HSPG core protein was diluted 1:250 and reacted with total E9 chick brain protein (100 µg) left untreated (lane 1), or treated with nitrous acid (lane 2), and immunopurified 6D2 HSPG (1 µg) left untreated (lane 3). Protein samples were separated on 4-15% gradient gels and transferred to nitrocellulose prior to immunoblotting with the anti-core protein antiserum.
Figure 6:
Western blots showing that anti-agrin
antibodies recognize a heparan sulfate proteoglycan from chick brain.
The blots were stained with a polyclonal antibody to the recombinant
agrin protein C-terminal fragment named CBA-1 (-agrin),
the 6D2 mAb (6D2), and an antiserum to the pPG5 agrin fusion
protein (
-pPG5). Lane 1, crude brain homogenate
from E10 chick embryos. Lane 2, proteins eluted with 1.5 M NaCl from Q-Sepharose. The Q-Sepharose had been loaded with E9
chick brain cytosol and was extensively washed with 0.5 M NaCl
and 4 M urea prior to elution. Lanes 3 and 7, vitreous humor protein from E10 embryos. Lanes 4 and 8, vitreous humor protein digested with heparitinase. Lanes 5 and 9, HSPG from E9 chick brain
immunopurified twice using the 6D2 and 3A12 mAbs. Lanes 6 and 10, immunopurified HSPG from E9 chick brain digested with
heparitinase. Lane 11, total protein from E9 chick brain left
untreated. Lane 12, total E9 chick brain protein digested with
nitrous acid. Lane 13, immunopurified HSPG from E9 chick brain
left untreated. The immunoblots shown in lanes 1-10 were
analyzed using 3.6-14% gradient polyacrylamide gels, with
molecular weight standards shown at the left. The immunoblot shown in lanes 11-13 is from a 4-15% gradient gel, with
molecular weight standards shown to the right. Note that the
agrin from brain and vitreous humor appears as a smear with a molecular
mass exceeding 400 kDa. This smear is reduced after heparitinase or
nitrous acid treatment to a narrow band of 250 kDa. The agrin can also
be enriched by binding to an anion exchange column (lane 2).
The antisera to agrin recognize the HSPG and its core protein
immunopurified from brain using the 6D2 and 3A12 mAbs, thus showing
that the two antigens are identical. Note also that the anti-agrin
antisera exhibit an identical reactivity with chick brain protein and
the 6D2 HSPG, when compared to the anti-HSPG core protein antiserum
(see Fig. 4).
As shown in Fig. 3, by both silver staining and
immunoblotting the HSPG and 220-kDa contaminant are detectable, raising
the possibility that these two proteins may be immunologically related.
We also found that by separating nitrous acid-treated HSPG core protein
on a 5% polyacrylamide gel, it was possible to resolve these two
proteins to the point of permitting excision of only the HSPG core
protein from the gel. The gel-purified core protein was then
electroeluted and injected into rabbits to generate a polyclonal
antiserum. This antiserum is specific for agrin, as determined by
immunoblotting of total chick brain protein with or without nitrous
acid treatment to eliminate HS chains (Fig. 4). This antiserum
was used to screen 250,000 recombinants from the random-primed E9 chick
brain cDNA library, and as shown in Fig. 1, 4 cDNAs were
isolated that were also found to be identical to chick agrin. One of
these clones that was identified with the anti-core protein antiserum
was also employed in Northern analyses, and reacts with the same 10-kb
agrin mRNA in embryonic chick brain (Fig. 2B). Although
the exposure of the Northern blot with the pPG12 clone was of shorter
duration than the pPG5 blot, it is evident that both cDNAs recognize a
mRNA that is expressed at higher levels in E9 chick brain than in E18
chick brain. The blot that was probed with the pPG12 clone was
previously probed with a 500-base pair mouse -actin DNA fragment,
prior to being stripped and probed with the pPG12 cDNA. As shown in Fig. 2B, these data demonstrate that only the agrin
mRNA levels are reduced from E9 to E18 of chick brain development. Most
important, these cloning data provide strong evidence that the agrin
cDNA encodes a HSPG in chick brain, since only agrin cDNAs were
identified following screening of the expression cDNA library with
various anti-HSPG antibodies.
Figure 5: Antibodies to the 6D2 HSPG recognize an agrin fusion protein. Clone pPG5 was used to generate a fusion protein as described under ``Experimental Procedures,'' and either 10-µg aliquots of protein from the soluble fraction of washed inclusion bodies of uninduced (lane 1) or induced (lane 2) E. coli, or 2.5 µg of purified inclusion body protein from induced cells, were separated on a 7.5% polyacrylamide gel, and immunoblotted with the polyclonal antiserum to the HSPG core protein (lanes 1 and 2) or the 6D2 mAb (lane 3).
To confirm that the
agrin HSPG is encoded by the agrin gene, a rabbit polyclonal antiserum
to an agrin protein expressed by stably transfected 293 HEK cells was
generated. The agrin used for antibody production was derived from the
well described CBA-1 C-terminal fragment of
agrin(25, 45) , which has been shown previously to
contain the AChR clustering activity of agrin(45) . This
anti-agrin antiserum was used to detect agrin in brain and vitreous
body by Western blotting. The antiserum detected agrin as a broad band
of over 400 kDa that shifted to a narrow band of 250 kDa after
treatment with heparitinase (Fig. 6, lanes 1-6).
Our experiments also show that agrin binds tightly to Q-Sepharose and
elutes from this anion exchange matrix only at ionic strength above 0.5 M NaCl (Fig. 6, compare lanes 1 and 2). Anion exchange chromatography is therefore a convenient
means of enriching for agrin from crude brain extracts. Using
Q-Sepharose chromatography, agrin can also be enriched from extracts of
chick embryo bodies excluding the brain. ()
The staining pattern of the recombinant agrin protein antiserum was indistinguishable from the 6D2 mAb (Fig. 6, lanes 7-10), thus confirming that the agrin gene codes for a HSPG in developing chick brain. We have also generated a polyclonal antiserum to the pPG5 agrin fusion protein, with this antiserum also showing reactivity with the same HSPG in E9 brain tissue and with the purified intact HSPG (Fig. 6, lanes 11-13). Again, the HSPG recognized by this antiserum appeared as a 400-kDa smear that is shifted to 250 kDa following nitrous acid treatment.
Figure 7: Fluorescent micrographs showing the distribution of brain-derived HSPG recognized by the 6D2 mAb and agrin. Adjacent sections through retina (R), optic nerve (ON), and optic chiasm (CH) of an E10 chick head were stained with the 6D2 mAb (a) and a polyclonal antiserum to the CBA-1 agrin protein fragment (b). The staining pattern of the optic nerve and retina, with the optic fiber layer (arrow) and the inner plexiform layer (arrowhead) exhibiting staining, are identical. D, diencephalon. Bar: 250 µm.
Finally, the identity
between the agrin HSPG and agrin was confirmed by examining expression
of the 6D2 HSPG in adult muscle, using the 6D2 mAb. A defining feature
of agrin is its expression at the synaptic sites of the neuromuscular
junction, and its co-localization with ACh receptors(37) .
Using the 6D2 mAb to the core protein of the HSPG, we show that the
HSPG is localized to the neuromuscular junction synaptic site, as
evidenced by co-localization with -bungarotoxin binding sites,
with lower levels of expression at extra-synaptic sites (Fig. 8). A similar distribution of the 6D2 HSPG has been
observed in embryonic pectoralis muscle. (
)This expression
pattern in muscle is identical to that of agrin(37) , providing
additional support for the conclusion that agrin is a nervous system
HSPG.
Figure 8:
The
6D2 HSPG is localized to the synaptic site of the developing
neuromuscular junction. Adult chick eye muscle (musculus obliquus
superior) was incubated with TRITC -bungarotoxin (A) or
6D2 mAb and fluorescein-conjugated anti-mouse secondary antibody (B), and visualized by epifluorescence microscopy. Expression
of the HSPG can be observed at the synaptic site of the neuromuscular
junction, as well as at extrasynaptic sites. The asterisk indicates a blood vessel. Bar: 10
µm.
The studies described herein were initiated in order to
obtain further information about a prominent HSPG from chick brain
which exhibits a developmentally-regulated pattern of
expression(23, 24) . Surprisingly, our cloning studies
have shown that the 6D2 HSPG core protein is identical to agrin, and
hence that agrin is a HSPG. This conclusion is supported by several
experimental observations, which include the demonstration that
antibodies to the HSPG recognize both agrin and the HSPG, that the
various HSPG antibodies only identified agrin cDNAs following
expression screening, that antibodies to agrin or an agrin fusion
protein exhibit imunoblotting patterns identical to the HSPG
antibodies, and that the 6D2 mAb to the HSPG stains the synaptic site
of the chick neuromuscular junction. In addition, we show that a
polyclonal antiserum to agrin reacts with a high molecular weight
heterogeneous protein smear by immunoblotting, which is typical for
proteoglycans. The agrin smear is shifted to a 250-kDa protein after
heparitinase treatment, in agreement with earlier studies using the 6D2
mAb to the 6D2 HSPG(23) . Agrin also binds tightly to anion
exchange matrices, showing that the molecule is highly negatively
charged, a hallmark of all proteoglycans. The core protein appears to
contain additional carbohydrate, as determined by
[H]glucosamine labeling(24) , but does
not contain other classes of GAG chains(23, 24) .
These data therefore show that agrin is the core protein of a large
HSPG. Lastly, studies carried out which have examined bovine kidney
basement membrane HSPGs suggested that tryptic fragments of a kidney
HSPG core protein exhibited high homology to rat agrin(46) ,
providing additional support for our conclusion that agrin is a HSPG.
It should be noted that it was previously believed that agrin cannot
be isolated as an intact molecule, and thus proteolytic fragments of
the protein were utilized in earlier characterizations of
agrin(47) . Also, previously published Western blots using
anti-agrin antisera show only agrin fragments of about 100 kDa in
chick(48) , but never show the intact agrin protein. Likewise,
in rat agrin has been identified by immunoblotting as a protein
slightly smaller than 200 kDa(27) . Like these previous
studies, when we use single percentage gels (i.e. 6%) to
analyze agrin expression in chick brain, we only detect agrin following
elimination of HS chains from the molecule (see Fig. 3).
However, when using gradient gels to analyze agrin in brain, our data
clearly show that agrin can be isolated either from brain or vitreous
body, and is readily detectable by Western blotting in crude extracts
of brain, optic nerve, or vitreous body. In our blots, the most
abundant agrin signal is that of the intact proteoglycan, and an
immunologically related 220-kDa contaminant and smaller fragments are
only minor contaminants. An explanation for the failure to previously
detect and isolate agrin may therefore lie in the fact that the intact
proteoglycan, with a molecular mass in excess of 400 kDa, is too large
to enter even low percentage gels and may therefore have never been
detected in these earlier studies. Our most recent experiments also
show, by immunoblotting with the antiserum to the chick agrin core
protein, that agrin in mouse and cow brain exists as a proteoglycan of
over 400 kDa, with a 250-kDa core protein. ()
Our demonstration that agrin is a HSPG that is expressed primarily during early periods of brain development is of interest, since the most prominent expression of agrin is in brain (25; our studies here), but the function of agrin in brain has remained elusive. Recent studies have suggested that brain agrin may be involved in synaptogenesis in the developing central nervous system, since it is expressed by various neuronal cell types in brain(29, 30, 44) . Brain agrin has also been proposed to participate in cell adhesion processes since it is secreted by axons along their pathway of growth (49) and contains laminin-like domains that could possibly function in cell adhesion(25, 27) . Our studies reported here therefore provide strong support for a proposed role of brain agrin in cell adhesion. We have shown previously that NCAM function is modulated by HSPGs (18, 21) and that the HSPG (agrin) is capable of binding to the heparin-binding domain of NCAM(24) . Our recent studies have also demonstrated that purified agrin HSPG can regulate adhesion to a functional domain of NCAM, and most importantly, that the HSPG can serve as a substrate for neural cell attachment(24) . With the demonstration here that the agrin gene encodes this HSPG, it is clear that agrin function in brain may range from promoting synaptogenesis to regulating cell adhesion mediated by heparin-binding proteins in the central nervous system that include NCAM, thrombospondin, laminin, and myelin-associated glycoprotein. In addition, adhesion proteins such as NCAM co-localize with agrin to the synaptic sites of the neuromuscular junction(50, 51) , raising the possibility that NCAM and agrin are interactive partners that regulate synaptogenesis during nervous system development.
Our demonstration that agrin is a HSPG is of particular interest in view of previous studies which have shown that heparin inhibits the ability of agrin to cluster ACh receptors(34, 35, 36) . These studies have shown that agrin-mediated induction of AChR clustering at the neuromuscular junction is inhibited by soluble heparin or heparan sulfate, with the proposal that agrin interacts with a cell surface HSPG at the neuromuscular junction(35) . Muscle cell lines deficient in heparan sulfate biosynthesis were also unable to form AChR aggregates in response to exogenous agrin, again suggesting that a HSPG is critical to agrin function(36) . Although these findings have been interpreted as indicating that agrin may interact with a HSPG, with this interaction critical to the AChR clustering activity of agrin, our data presented here could also be consistent with heparin inhibiting agrin function because of agrin's heparan sulfate chains being required for its function. However, since GAG-deficient muscle cells do not cluster AChRs in response to exogenous agrin, these data suggest that the muscle cell is providing the HSPG critical to agrin function. Although it remains possible the proteoglycan form of agrin in muscle is needed for agrin-induced AChR aggregation, it is also possible that a muscle HSPG distinct from agrin is necessary for agrin activity. However, because we have recently shown that agrin can promote cell-substratum adhesion, with this function dependent on its heparan sulfate(24) , it is apparent that future studies will be necessary to elucidate the possible role of agrin's heparan sulfate chains in AChR aggregation and synaptogenesis.
Recent
studies involving agrin have emphasized identifying the receptor for
this extracellular protein, since this would further one's
understanding of mechanisms underlying the function of agrin.
Preliminary studies showed that the agrin receptor is localized to the Torpedo electric organ postsynaptic membrane, consistent with
agrin's role in synaptogenesis(52) . Recent studies have
implicated -dystroglycan as a putative agrin
receptor(31, 32, 33) , with this protein
being distributed over the entire muscle surface, including the
postsynaptic membrane. Our studies provide evidence that NCAM may also
function as an agrin-binding protein, since immunopurified agrin is
capable of binding to the heparin-binding domain of NCAM (24) and antibodies to NCAM partially inhibit cell adhesion to
an agrin substratum(24) . Thus, since NCAM and agrin
co-localize at the synaptic site of the neuromuscular
junction(50) , it is of interest to consider the possibility
that these two proteins may participate as binding partners in the
regulation of synaptogenesis. One can also speculate that additional
agrin-binding proteins may also exist in view of the presence of
heparan sulfate on the protein, and therefore a multitude of
heparin-binding proteins can serve as putative agrin-binding proteins.
With this capability of serving as a multifunctional protein, agrin may
have the potential to regulate the function of a diverse population of
nervous system proteins, and thus future studies will have the
opportunity to explore the function of agrin in a variety of
developmental processes.