From the
A novel agrin isoform was identified based on the isolation of
an agrin cDNA from E9 chick brain that lacked 21 base pairs (bp) in the
NH
Critical to the proper development of the nervous system is a
highly regulated pattern of gene expression that defines processes
necessary for the interaction of cells with their environment and
neighboring cells. Numerous studies have documented the importance of
extracellular signals in neural development, with these signals
regulating processes as diverse as cell-matrix interactions, cell
motility, and cell differentiation. A well-characterized model system
for the study of the role of extracellular matrix proteins in nervous
system development is agrin and its association with neuromuscular
synaptogenesis (for reviews see Refs. 1 and 2). Agrin was initially
identified as a neuronal basal lamina protein capable of inducing the
aggregation of acetylcholine receptors (AChR)
Recent studies have focused on a molecular characterization of agrin
mechanisms and have shown that agrin has alternatively spliced mRNA
isoforms(12, 13, 14, 15) . The
demonstration that alternative splice sites, designated A and B, are
restricted to the region of the mRNA that codes for the functional
COOH-terminal domain of agrin has been of particular
interest(12, 14) , since these data indicate that the
function of agrin can be regulated in a cell- or tissue-specific
manner. For example, it is clear that the agrin
Our recent studies demonstrating that agrin is a
HSPG are noteworthy, since we have shown previously that this HSPG is
capable of binding to and modulating NCAM function(21) . In view
of the demonstration that the majority of agrin expression occurs in
brain(8, 22) , but agrin's function in brain has
remained elusive, our studies raise the interesting possibility that
brain agrin has a role in cell adhesion processes. Accordingly, our
studies suggest that the heparan sulfate chains of agrin, which appear
to be localized to the NH
For PCR analysis at different developmental ages, the
use of equivalent amounts of cDNA was confirmed by RT-PCR of cDNA using
chicken
We demonstrate in the present study that an additional
spliced isoform of agrin is expressed in brain and non-neuronal
tissues. However, unlike the previous identification of alternatively
spliced isoforms of agrin (12, 14) where the splice
sites were restricted to the region of the agrin mRNA encoding the
COOH-terminal domain of the protein, our studies indicate a novel
splice site in the 5`-region of the agrin mRNA (Fig. 5). It is
therefore interesting to note that in previous studies on the agrin
gene in rat the 5`-region of the gene was not mapped, and thus the
presence of this alternatively spliced exon had gone
undetected(26) . Our data strongly suggest that in brain this 21
bp insertion into chick agrin appears to be localized to neuronal
cells, since analysis of chick astrocyte mRNA by RT-PCR indicates the
presence of agrin mRNA lacking the 21-bp domain. Upon analysis of
non-neuronal chick tissues, in particular smooth and cardiac muscle, we
have also observed an abundance of the agrin isoform lacking the
alternatively spliced 21-bp insert. It is presently unclear why the
agrin isoform containing the 21-bp insert is detected in chick glia and
muscle, although it is likely that slight neuronal contamination of the
astrocyte cultures can account for the minor levels of agrin mRNA
containing the 21-bp exon in these cultures(25) . It remains to
be determined whether non-neuronal cell populations in gut and heart
are expressing the spliced form of agrin, or if neuronal components
such as cardiac neural crest account for this expression. However, it
has been shown previously that the low levels of the mRNA encoding the
agrin
It is
presently unclear what function the alternative splicing of the 21-bp
exon may impart on agrin, especially in light of previous studies which
have shown that the splicing out of alternative exons in non-neuronal
agrin results in an inability to induce AChR aggregation (12, 14, 28).
It is of interest that the alternatively spliced exon we have
identified is localized to a region of agrin which has been suggested
to contain the signal sequence for this protein. Thus, splicing out of
the 21-bp exon at splice site C would affect the putative signal
sequence, conceivably regulating the processing of this protein in
non-neuronal and neuronal cells. For example, the splicing out of the
21-bp exon removes 7 amino acids from the putative signal sequence of
agrin, including the consensus signal peptidase site immediately
preceding the putative first amino acid of the mature protein (Fig. 1). Thus, with this signal peptidase site deleted, two
possibilities exist in terms of processing of this splice variant of
agrin: 1) the next available signal peptidase site would be utilized,
resulting in non-neuronal cells lacking 24 amino acids at their
NH
Since in previous studies it was also thought that agrin could not
be isolated in its intact form(3) , the NH
We thank Drs. Jeff Masters, John Oberdick, and
Pappachan Kolattukudy for their helpful discussion during the course of
this work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal encoding region of the agrin mRNA. Reverse
transcription-polymerase chain reaction (RT-PCR) of E9 chick brain mRNA
confirmed the existence of this agrin isoform in brain, although the
novel splice variant represents a minor fraction of agrin mRNA in
brain. However, upon analysis of chick brain astrocyte mRNA, smooth
muscle mRNA, and cardiac muscle mRNA by RT-PCR, we show that this novel
agrin isoform is the predominant agrin isoform in these non-neuronal
cell populations. We extended our analyses to examine the expression of
this agrin mRNA isoform during chick development and show that the
agrin mRNA lacking this 21-bp exon is up-regulated with brain
development, consistent with the increase in glial number during brain
development, while the agrin isoform that does not undergo splicing and
thus contains the 21-bp exon is down-regulated in brain development.
Because the 21-bp exon is inserted in the region of chick agrin which
encodes the putative signal sequence of agrin, with the signal
peptidase site immediately preceding the putative first amino acid of
the mature protein being deleted as a result of splicing, these data
raise the interesting possibility that the presence or absence of this
alternatively spliced exon may differentially regulate processing of
the agrin protein in neuronal and non-neuronal cells, respectively.
(
)in the developing neuromuscular
junction(3, 4) , with its ability to also induce the
aggregation of other synapse-associated proteins suggesting a key role
for agrin in synaptogenesis(5, 6) . Although it was
believed that agrin could not be isolated in its intact form from basal
lamina(3) , it was recognized that an agrin proteolytic fragment
of about 100 kDa was capable of mediating the AChR clustering effects
of agrin(3, 7) . However, recent studies have
demonstrated that the agrin mRNA encodes a 400-kDa heparan sulfate
proteoglycan, which contains an approximately 250-kDa core
protein(8) . In light of the wealth of data implicating a role
for heparin and/or heparan sulfate proteoglycans in agrin
function(9, 10, 11) , it remains to be
determined if heparan sulfate proteoglycans other than agrin are
involved in AChR aggregation during neuromuscular synaptogenesis.
isoform,
which lacks the alternative exons, is not functional in AChR clustering
assays(12, 14) . In addition, single-cell RT-PCR
experiments have shown convincingly that the agrin
isoform is the predominant isoform expressed by non-neuronal
cells, with combinations of alternative splicing of exons occurring in
neurons(16) . Thus, these data imply that functional forms of
agrin, defined in terms of their ability to aggregate AChR, are
restricted to neurons. However, the role of alternative splicing in
agrin functions becomes clouded, based on recent studies describing the
role of
-dystroglycan as a putative receptor for
agrin(17, 18, 19, 20) . The binding of
agrin to
-dystroglycan occurs in a heparin-dependent
manner(17) , consistent with earlier analyses of agrin function,
but the agrin-dystroglycan interaction is not
isoform-dependent(20) . Likewise, the ability of
agrin-dystroglycan interactions to aggregate AChR in muscle is not
inhibited by antibodies that perturb agrin-dystroglycan binding (20).
Thus, it remains to be determined by what mechanism(s) alternative
splicing of the agrin mRNA regulates the expression of functional forms
of the protein.
-terminal half of the molecule,
are critical to cell adhesion to NCAM(8, 21) , but are
not likely involved in the AChR clustering activity of agrin. In the
course of our molecular cloning of agrin, we isolated a cDNA lacking 21
bp in the corresponding 5` region of the mRNA. In the present study, we
show by RT-PCR that splicing of this novel exon occurs in a cell- and
tissue-dependent fashion, with splicing occurring predominantly in
non-neuronal cells. Thus, in accord with the studies of Smith and
O'Dowd(16) , it appears that the inclusion of
alternatively spliced exons in agrin mRNA is preferentially restricted
to neurons. We have named this new agrin isoform in non-neuronal cells
agrin-related protein-3 (ARP-3), and the alternative splice site C, in
keeping with the previous nomenclature for agrin isoforms in
chicken(14) .
Isolation of Agrin cDNA
A cDNA encoding agrin,
and lacking 21 bp when compared to the previously published chick agrin
cDNA(14) , was isolated by screening a random-primed E9 chick
brain cDNA library with a restriction fragment from a previously
isolated agrin cDNA(8) . Briefly, a 711-bp SstI
restriction fragment from the 5` region of agrin clone pPG3 (8) was used to screen 600,000 recombinants from the E9 chick
brain cDNA library according to published protocols (8). One resulting
agrin clone, named 801, was found to lack 21 bp when compared to the
published chick agrin nucleotide sequence(14) .
RT-PCR Analysis of ARP-3 mRNA Expression
Total RNA
from various ages of embryonic chick brain and heart, or from cultured
chick brain astrocytes, was isolated according to Chomczynski and
Sacchi (24). Poly(A) RNA was then isolated using the
Straight A's mRNA isolation system (Novagen). First strand cDNA
was then synthesized from 1 µg of mRNA using the reverse
transcriptase SuperScript (Life Technologies, Inc.). RNA was then
hydrolyzed with NaOH at 65 °C for 30 min, and, following
neutralization with acetic acid, the cDNA was purified using Geneclean
(Bio 101). Following isolation of the cDNA by precipitation with
glycogen and ethanol, 10% of the isolated cDNA was used for PCR
amplification.
-actin PCR primers. For PCR amplification, 1 µl of the
cDNA synthesis mixture described above was used. Primer pairs used for
RT-PCR of agrin mRNA were a forward 19-mer: 5`-TGGAGCACTGCGTGGAAGA-3`
and a backward 20 mer: 5`-TGCAGACACAGGAGGCTTGG-3`. cDNA was amplified
for 28 cycles at an annealing temperature of 52 °C, and PCR
products were then analyzed by electrophoresis on 3% agarose gels, with
DNA visualized by ethidium bromide staining. To confirm the
authenticity of the PCR products, they were subcloned into the
pBluescript vector pCR-Script SK(+) (Stratagene) and subsequently
sequenced by the dideoxy chain termination method(23) . The
lower M
PCR product was shown to contain 146 bp
and lack the 21 bp insertion, while the larger PCR product was 167 bp
in length and contained the 21-bp insertion.
Identification of Novel Agrin mRNA Splice
Variant
During the course of our molecular cloning of an
NCAM-binding HSPG, which was subsequently shown to be identical to
agrin(8) , we isolated an agrin cDNA that exhibited sequence
differences to the published chick agrin cDNA(22) . This partial
cDNA, named clone 801, was isolated by rescreening of a random-primed
E9 chick brain cDNA library with a restriction fragment from agrin
clone pPG3, which encoded nucleotides 560-1271 of agrin(8) .
Upon sequence analysis, it was determined that clone 801 was identical
with the published chick agrin cDNA at its 5` and 3` ends, but a 21-bp
region from nucleotides 169-189 was absent from clone 801 (Fig. 1). Since these data raised the possibility that this cDNA
defines a new splice variant of agrin, we named this cDNA agrin-related
protein-3 (ARP-3), and the alternative exon encoding this domain was
named C, in accord with the previous nomenclature for alternative
splice sites in chicken agrin(14) .
Figure 1:
Comparison of nucleotide and deduced
amino acid sequences for agrin and ARP-3 cDNAs. The nucleotide position
is indicated at top left, with the corresponding amino acid
sequence shown at the bottom. The underlined nucleotide sequence denotes the forward primer sequence used for
RT-PCR studies. The dashed line in the ARP-3 sequence
indicates the absence of the 21-bp insertion observed in the previously
published chick agrin cDNA. The double-underlined amino acid
sequences represent consensus signal peptidase sites in the
NH-terminal region of agrin, showing that the first signal
peptidase site would be eliminated by alternative
splicing.
In order to confirm
whether the ARP-3 cDNA was present in chick brain as an mRNA, we
designed PCR primers flanking this region. The forward primer sequence
is indicated in Fig. 1, and the reverse primer was from
nucleotides 319-300 of chick agrin. As shown in Fig. 2, by
PCR of the clone 801 plasmid, a 146-bp product is obtained as predicted
by the cDNA sequence. Upon PCR amplification of E9 chick brain mRNA
that had been reverse-transcribed to yield single strand cDNA, the
predominant product obtained was a 167-bp fragment, with the 146-bp
product representing a minor mRNA species in E9 chick brain (Fig. 2). The authenticity of these PCR products was confirmed by
subcloning of the products into PCR pBluescript, followed by dideoxy
sequencing. The nucleotide sequence of each PCR product confirmed that
the 167-bp fragment is identical with the published chick agrin cDNA,
and that the 146-bp fragment is identical with the ARP-3 cDNA.(
)Thus, these data show that a novel agrin mRNA species
is expressed in chick brain, albeit at extremely low levels.
Figure 2:
RT-PCR analysis of E9 chick brain agrin
mRNA expression. mRNA was isolated from E9 chick brain,
reverse-transcribed with SuperScript reverse transcriptase and
amplified by PCR using primers flanking the 21-bp region not inserted
into ARP-3. PCR products were then separated on 3% agarose gels, with
DNA visualized by ethidium bromide staining. Lane 1, positive
control using clone 801 plasmid, showing that a 146-bp PCR product is
obtained; lane 2, PCR amplification of E9 chick brain cDNA,
showing the presence of a major 167-bp product, as well as the 146-bp
product corresponding to the ARP-3 cDNA; lane 3, negative
control using E9 chick brain mRNA not treated with reverse
transcriptase, showing absence of any PCR products; lane 4,
negative control showing absence of PCR products when E9 brain cDNA is
omitted from the PCR reaction mixture.
Tissue Distribution of ARP-3 mRNA
Since previous
analyses of agrin mRNA expression have suggested that non-neuronal cell
agrin mRNA is subjected to alternative splicing, usually resulting in
splicing out of all identified alternatively spliced exons, with only
various combinations of splicing occurring in neurons(16) , we
were interested in ascertaining whether the alternatively spliced ARP-3
mRNA is also restricted to non-neuronal cells. To address this
question, mRNA was isolated from chick brain astrocyte cultures, E10
gut (smooth muscle), and E10 heart (cardiac muscle). Following reverse
transcription, these cDNAs were amplified using the PCR primers that
flank the 21-bp region absent from ARP-3. These data show that the
major PCR product in glia is 146 bp, indicating that the primary
transcript in chick glia is the ARP-3 mRNA, lacking the 21-bp insertion (Fig. 3). In addition, both muscle tissues show an enrichment for
the ARP-3 mRNA, as exhibited by the abundant signal for the 146-bp PCR
product (Fig. 3). Although it is also evident that the 167-bp PCR
product is obtained using muscle and glia mRNA, it remains to be
determined what cellular source is responsible for expression of the
unspliced form of agrin (i.e. containing the 21-bp insertion).
Figure 3:
RT-PCR analysis of agrin isoform mRNA
expression in embryonic chick brain, glia, and muscle. mRNA was
isolated from E9 chick brain, cultured chick brain astrocytes, E10
smooth muscle (gut), and E10 cardiac muscle, and subjected to RT-PCR. brain, E9 chick brain, showing that the predominant agrin
isoform contains the 21-bp insertion; glia, chick astrocytes,
showing that the major agrin isoform lacks the 21-bp sequence and
corresponds to ARP-3; gut, E10 smooth muscle cDNA after PCR,
with this tissue exhibiting significant amounts of both isoforms,
although the ARP-3 isoform predominates; heart, E10 chick
heart, showing the presence of both agrin
isoforms.
Developmental Expression of Agrin Isoforms
To
extend our understanding of the expression pattern of the ARP-3 agrin
mRNA, we analyzed by RT-PCR the expression of this mRNA during chick
brain and heart development. Based on our demonstration that glia
exhibit an enrichment for the ARP-3 mRNA, we predicted that its
expression should become augmented during brain development, consistent
with gliogenesis during late development. As shown in Fig. 4A, during chick brain development the ARP-3 mRNA
does display an increase in abundance, while the major agrin mRNA
species, containing the 21-bp insertion, is diminished. In parallel
analyses, we employed -actin primers for RT-PCR, and these data
indicate that different cDNA levels did not account for the observed
alteration in agrin expression by RT-PCR, as similar levels of actin
could be detected at each developmental age (Fig. 4).
Figure 4:
Analysis of developmental expression of
agrin isoforms by RT-PCR. A, E9 and E18 chick brain mRNA was
analyzed by RT-PCR, followed by separation of PCR products on 3%
agarose gels. It can be seen that while the major brain agrin isoform
is diminished with development, the level of the ARP-3 agrin isoform is
augmented. B, E7-E18 chick heart mRNA was analyzed by RT-PCR,
followed by agarose gel electrophoresis. These data show a significant
diminution of both agrin isoforms occurring subsequent to E10 of chick
heart development. In both brain and heart cDNAs, -actin levels
were determined by RT- PCR, and these data show that the 340-bp
-actin PCR product is expressed at similar levels during
development.
We also
examined by RT-PCR the expression of agrin mRNA isoforms during chick
heart development, and these data indicate that both agrin isoforms are
reduced during heart development (Fig. 4B). The pattern
of expression of these isoforms is similar in chick heart, with both
species displaying a decrease in abundance subsequent to E10 of heart
development. Thus, it appears that distinct non-neuronal cell
populations display differences in expression of the ARP-3 agrin
isoform, with glial expression in brain resulting in a developmental
up-regulation, while in heart this isoform is down-regulated during
development.
isoform can be expressed in muscle and
non-neuronal tissues(14, 16, 27, 28) ,
indicating that not all alternative exons are spliced out in
non-neuronal agrin. Thus, it remains possible that the splicing at site
C is regulated in non-neuronal cells. Although this remains to be
confirmed, our data do suggest that in non-neuronal cells the majority
of agrin isoforms lack the C exon, and thus it appears that the
prevalent agrin isoform in non-neuronal cells is
agrin
.
Figure 5:
Schematic model of agrin, showing
structural domains and alternatively spliced exons. The alternative
splice sites are indicated with their corresponding amino acid
sequences and show that the site C splice site is contained within the
putative signal sequence peptide of agrin. -, putative
signal sequence;
, Kazal type protease inhibitor; &cjs2090;,
laminin homology domain;
, epidermal growth factor-like repeat;
°°°&cjs0822;, potential GAG attachment site;
&cjs0822;, potential N-linked glycosylation site;
/&cjs0822;&cjs0822;, alternative splicing
site.
Based on our observation that in chick brain
the predominant agrin form in glia lacks the 21-bp insert at splice
site C, we reasoned that with development we should see an augmentation
in expression of this isoform, concomitant with gliogenesis. Since
previous studies have shown a pronounced diminution in agrin during
chick brain development (8, 22), this would raise the possibility that
glial agrin could be mediating distinct functions as neuronal agrin is
down-regulated. Our present studies demonstrate a significant increase
in the expression of ARP-3 mRNA, coincident with a marked decrease in
the unspliced form of agrin. Thus, these data indicate distinctive
patterns of regulation of agrin during brain development in neurons and
glia. In light of our recent demonstration that agrin is capable of
modulating NCAM function (21), one can speculate that up-regulated
glial expression of agrin during brain development may selectively
modulate glial and/or glial-neuronal cell interactions, by contributing
to localized increases in agrin expression during development.
terminus (see Fig. 1); 2) the character of the
signal sequence would be altered, possibly precluding its removal and
thus resulting in an agrin protein that would remain associated with
the plasma membrane, rather than being secreted directly. However, this
latter possibility would seem unlikely since agrin has been shown to be
a secreted protein in both neurons (29, 30) and
glia(21) , although our analyses in glia used long-term
metabolic labeling of cultures which may have masked cleavage of a
membrane-associated protein, with its subsequent release into the
conditioned medium(21) . Although we have not yet been able to
elucidate the functional significance of the splicing at site C, an
alternative possibility that must be considered is that the putative
first amino acid in the mature agrin protein (Asp
) is in
fact not the NH
terminus of the mature protein, and thus
the putative signal sequence upstream of this amino acid is not
agrin's signal peptide. This possibility warrants consideration,
since the absence of splicing would then result in the insertion of 7
amino acids in the mature protein which could possibly modulate the
structure of the protein. For example, the 7-amino acid insertion
contains a cluster of basic amino acids, which could regulate the
ability of agrin to interact with anionic molecules. Likewise, the
7-amino-acid insert could introduce a putative protease cleavage site
that could then alter the structure of the protein containing the
insert. Moreover, in view of recent studies which have shown that agrin
encoded by the full-length agrin cDNA is not secreted, and that the
signal peptide of hemagglutinin had to be employed to facilitate
secretion of the full-length agrin protein(28) , it remains
possible that the signal sequence of agrin has not been identified.
-terminal
amino acid of the mature protein has never been conclusively
identified. We therefore undertook protein sequencing experiments using
immunopurified agrin from chick brain, which we have shown can be
isolated intact as a HSPG(8, 21) . The heparan sulfate
chains were removed from this agrin using heparitinase, and the protein
was then separated by gel electrophoresis and transferred to a
polyvinylidene difluoride membrane. Unfortunately, the
NH
-terminal amino acid sequence could not be obtained from
this preparation, or from soluble purified agrin, suggesting that the
NH
terminus is blocked.
It therefore remains to
be determined if splicing of this 21-bp exon alters the properties of
the mature protein versus the signal peptide. Thus, our goal
in future studies will be to elucidate the possible function of this
splicing event in agrin, with a focus on whether the processing of
agrin is affected by splicing at site C and whether this splicing may
modulate the ability of agrin to interact with adhesion proteins, such
as NCAM, or its ability to regulate AChR aggregation in muscle.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.