From the Department of Preclinical Veterinary Sciences, University
of Edinburgh, Edinburgh EH9 1QH, United Kingdom
Periaxin was first described as a 147-kDa protein
that was suggested to have a potential role in the initiation of myelin deposition in peripheral nerves based upon its abundance, cell type specificity, pattern of developmental expression, and localization (Gillespie, C. S., Sherman, D. L., Blair, G. E.,
and Brophy. P. J. (1994) Neuron 12, 497-508). Here we
show that the murine periaxin gene spans 20.6 kilobases and encodes two
mRNAs of 4.6 and 5.2 kilobases that encode two periaxin isoforms,
L-periaxin and S-periaxin of 147 and 16 kDa respectively. The larger
mRNA is produced by a retained intron mechanism that introduces a
stop codon and results in a truncated protein with an intron-encoded C
terminus of 21 amino acids. Both proteins possess a PDZ domain at the N
terminus; nevertheless, they are targeted differently in Schwann cells. Like other proteins that contain PDZ domains, L-periaxin is localized to the plasma membrane of myelinating Schwann cells: in contrast, S-periaxin is expressed diffusely in the cytoplasm. This suggests that
proteins that contain this protein-binding module may also participate
in protein-protein interactions at sites other than the cell
cortex.
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INTRODUCTION |
Periaxin was first identified as a relatively abundant 146-kDa
protein of myelinating Schwann cells in a screen for novel cytoskeleton-associated proteins with a role in peripheral nerve myelination (1). Like P0, the major integral membrane
protein of peripheral nervous system myelin, periaxin is detectable at an early stage of peripheral nervous system development (2). However,
in contrast to P0, periaxin is not incorporated into compact myelin (1), but is initially concentrated in the plasma membrane, the abaxonal membrane (apposing the basal lamina), and the
adaxonal membrane (apposing the axon). As myelin sheaths mature periaxin becomes concentrated in the abaxonal membrane and plasma membrane (2). This shift in the localization of the protein in the
Schwann cell after completion of the spiralization phase of axon
ensheathment suggests that periaxin may participate in the
membrane-protein interactions that are required to stabilize the mature
sheath. To shed light on the protein's function, we were particularly
interested to determine if modular protein-binding domains might be
represented in the periaxin amino acid sequence. Although no such
domains were identified from initial data base comparisons (1), here we
report that the periaxin gene does encode one of the most interesting
of these protein-binding motifs to emerge over recent years, namely the
PDZ domain.
The PDZ domain was named after the three proteins in which it was first
identified, namely post-synaptic density protein
PSD-95, Drosophila
discs large (dlg) tumor
suppressor gene, and the tight junction-associated protein
ZO-1 (3). It consists of an approximately
90-amino acid protein-binding motif found in proteins that interact
with the cytoplasmic tail of plasma membrane proteins and with the
cortical cytoskeleton (4). Although the binding site for some PDZ
domains is the simple peptide sequence (S/T)XV found at the
C terminus of certain plasma membrane proteins (5), PDZ-containing
proteins can recognize somewhat different sequences and can even form
homophilic clusters with the PDZ domains of other proteins (6, 7) . So
far, two major functions have been ascribed to PDZ domains on the basis of their interactions with plasma membrane proteins and their presence
in known signaling molecules such as dlg (6). First, they
may organize and recruit proteins to the plasma membrane as has been
proposed for PSD-95 (3). Secondly, they may link transmembrane proteins
with the actin cytoskeleton via actin-binding proteins such as protein
4.1 (8-10).
Here we report the structure of the murine periaxin gene, which reveals
that there are two periaxin isoforms, L-periaxin and S-periaxin. The
smaller protein, S-periaxin, is generated by a relatively rare retained
intron mechanism (11), which is probably favored by the presence of
suboptimal 5'- and 3'-splice sites in the final intron together with a
downstream putative exonic splicing enhancer (12, 13). Although both
are PDZ proteins, L- and S-periaxin are targeted differently in the
Schwann cell, indicating that the PDZ domain is not the sole
determinant of their subcellular localization. To our knowledge this is
the first example of the differential localization of two protein
isoforms with the same PDZ domain.
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EXPERIMENTAL PROCEDURES |
Isolation and Characterization of Genomic Clones--
A 129SV
murine genomic library (gift from Dr W. Skarnes, Center for Genome
Research, Edinburgh University) in the bacteriophage Lambda Dash II
(Stratagene, Cambridge, UK) was screened by plaque hybridization (14)
with a 32P-labeled probe comprising nucleotides 1-3000 of
rat periaxin cDNA (1). Hybridization was at 60 °C in QuikHyb
(Stratagene, Cambridge, UK), and the filters were washed in 2 × SSC (300 mM NaCl, 30 mM sodium citrate, pH 7),
0.1% (w/v) SDS at room temperature and finally at 60 °C in 0.2 × SSC, 0.1% SDS. From 1 × 106 clones, twelve
positive plaques were identified after three rounds of screening, and
EcoRI fragments of each insert were subcloned into the
pIBI30 plasmid (IBI, Cambridge, UK) for analysis. The ends of each
clone were sequenced by the dideoxy chain termination method (15) using
a T7 DNA polymerase kit (Pharmacia LKB Biotech, Uppsala, Sweden), which
showed that the twelve clones comprised three groups of sequences that
overlapped (H1, F2, and I1). Oligonucleotide primers designed from
these sequences were used to determine the order of the
EcoRI fragments within the gene by
PCR1 using purified
bacteriophage DNA (1 µl of 1:500 dilution) as template (first cycle
of 94 °C for 5 min, 60 °C for 1 min, and then 72 °C for 1 min;
36 cycles of 94 °C for 1 min, 60 °C for 1 min, and then 72 °C
for 1 min, 7 min during the last cycle). The reactions (50 µl)
included primers (5 µM) and Dynazyme DNA polymerase (5 units) (Flowgen Ltd., Sittingbourne, UK). Periaxin sequence was mapped
to the EcoRI fragments by Southern blot with regional
periaxin probes. Briefly, EcoRI-digested plasmid DNA from
each EcoRI subclone was electrophoresed on a 1% agarose gel and vacuum blotted to Magma nylon membrane (Micron Separations Inc.,
Westborough, MA). The membrane was hybridized with
32P-labeled rat periaxin cDNA fragments covering the
entire rat cDNA, and the order of the genomic clones was
determined. The cDNA and genomic sequences were compared using the
University of Wisconsin GCG software package. Analysis of the three
clone types from the first screening revealed the absence of sequence corresponding to the first 297 bases of the rat cDNA, which
includes the initiation codon and the 5'-untranslated region. Therefore a further 6 × 105 plaques were screened with a probe
corresponding to nucleotides 50-297 of the rat sequence (1), which was
generated by reverse transcription-PCR of the 5'-end of mouse periaxin
mRNA (see below). Two clones were isolated and characterized as
described above and were shown to be identical. This sequence (HH1) did
not overlap with the 5'-end of H1. However HH1 and H1 were shown to be
contiguous by PCR performed on 129SV genomic DNA as template (first
cycle of 94 °C for 5 min, 60 °C for 1 min, and then 72 °C for
1 min; 36 cycles of 94 °C for 1 min, 60 °C for 1 min, and then
72 °C for 1 min, 7 min during the last cycle). The reactions (50 µl) contained genomic DNA (200 ng) primers (5 µM) and
Dynazyme DNA polymerase (5 units) (Flowgen Ltd.). The PCR product was
isolated from an agarose gel with the QIAEX II gel extraction kit
(Qiagen Ltd, Crawley, UK) cloned into the pGEM-T vector (Promega Ltd., Southampton, UK) according to the manufacturer's instructions and
sequenced using a T7 sequencing kit, with T7 and M13(R) primers (Pharmacia).
Reverse Transcription-PCR--
To obtain a probe for the 5'-end
of the mouse periaxin mRNA, total RNA was isolated from the sciatic
nerves of 15-day-old mice with RNAzol B (Biogenesis, Bournemouth, UK)
according to the manufacturer's instructions. Reverse transcriptions
(20 µl) contained RNA (2 µg), random hexamers (150 ng),
dithiothreitol (10 mM), dNTPs (0.5 mM), and
SuperScript reverse transcriptase (400 units) (Life Technologies Inc.,
Paisley, Scotland) in first strand synthesis buffer and were incubated
at 42 °C for 1 h. The reactions were terminated by heating at
80 °C, and 1 µl of this reaction was used as a template for PCR
using primers corresponding to nucleotides 50-71 and 631-650 of the
published rat periaxin cDNA sequence (1). PCR conditions were: five
cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min followed by 26 cycles of 94 °C for 40 s, 55 °C for 1 min, and 72 °C for 1 min (4 min during last cycle). Reactions (50 µl) contained primers (5 µM) and Dynazyme DNA
polymerase (1 unit) (Flowgen Ltd.). The product was subcloned into the
pGEM-T (Promega Ltd., Southampton, UK) vector according to the
manufacturer's instructions and sequenced with a T7 DNA polymerase kit
(Pharmacia).
Determination of Periaxin Transcriptional Initiation
Site--
An oligonucleotide complementary to nucleotides 50-71 of
the rat periaxin cDNA (1) was end-labeled with
[
-32P]ATP and T4 polynucleotide kinase. Total RNA (10 µg) from the trigeminal nerves of 13-day-old mice was hybridized to
labeled oligonucleotide (5 pmol) and reverse transcribed for 10 min at 80 °C followed by 18 h at 42 °C. Reverse transcription
employed SuperScript reverse transcriptase (Life Technologies Inc.) in a buffer supplied by the manufacturer and included 50 µg/ml
actinomycin D. Extended products were resolved on a 6% polyacrylamide
DNA sequencing gel adjacent to a dideoxy sequencing ladder comprising the 5'-end of periaxin primed using an oligonucleotide complementary to
nucleotides 103-122 of mouse periaxin cDNA.
Isolation of cDNA Clones Encoding Two Periaxin
Isoforms--
Two periaxin mRNAs of 5.2 and 4.6 kb were detected
in mouse and rat sciatic nerve. We had previously cloned the cDNA
for the smaller mRNA (1). To identify how the larger mRNA
differed from the 4.6-kb mRNA, 3 × 105 clones of
a 15-day-old rat sciatic nerve cDNA library constructed in
EcoRI-digested
gt11 (Promega Ltd., Southampton, UK)
according to the manufacturer's instructions were screened with a
probe comprising nucleotides 1-597 of the rat periaxin cDNA
previously described (1). Screening was carried out essentially as for the genomic library, and 24 positive plaques were purified. DNA from
each of these phage clones was digested with NotI, and the inserts were subcloned into the pGEM11zf plasmid (Promega Ltd., Southampton, UK) and sequenced. Of 11 clones that included a sequence homologous to exon 6 of the mouse gene, five included a sequence corresponding to the intron between exons 6 and 7. Total RNA from both
rat and mouse sciatic nerves was subsequently Northern blotted with a
PCR-generated probe for this intronic sequence with identical results.
Northern Blotting--
Total RNA from 15-day-old mouse
sciatic nerves was electrophoresed on 0.8% agarose formaldehyde gels
and transferred to Magna nylon membrane (Micron Separations Inc.,
Westborough, MA) by vacuum blotting in 20 × SSC. Filters were
probed for 1.5 h in Rapid Hyb buffer (Amersham) at 65 °C with
an exon 7-specific probe, a 1.3-kb restriction fragment corresponding
to nucleotides 1-1324 of the rat periaxin cDNA sequence, or a PCR
product homologous to the intron 6 of the mouse gene labeled with
[
-32P]dCTP by random priming (Life Technologies Inc.)
and were washed to a final stringency of 0.2 × SSC at 65 °C.
The intron probe was prepared using one of the five rat cDNA clones
isolated as described above as template with primers that flanked 400 bp of intron sequence. The PCR conditions used for the preparation of this intronic probe were: first cycle of 94 °C for 1 min, 55 °C for 1 min, and then 72 °C for 1 min; 28 cycles of 94 °C for
40 s, 55 °C for 1 min, and then 72 °C for 1 min, 4 min
during the last cycle. Reactions (50 µl) contained primers (5 µM) and Dynazyme DNA polymerase (0.5 unit) (Flowgen
Ltd.).
Antibody Production--
The anti-SPeri and anti-NTerm
antibodies were raised in rabbits against the synthetic peptides
AKLVRVLSPVPVQDSPSDRVAAAC and EARSRSAEELRRAEC, respectively, which were
generous gifts from Prof. N. Groome, Department of Biology, Oxford
Brookes University. The former peptide corresponded to the C-terminal
23 amino acids of mouse S-periaxin, and the latter comprised the
N-terminal 14 amino acids of mouse periaxin ,which is identical in
L-periaxin and S-periaxin. The C-terminal cysteine residue of each
peptide was coupled to Keyhole Limpet hemocyanin by standard techniques and used to immunize rabbits. Anti-SPeri antibody was affinity purified
by immunoabsorption to a column of peptide coupled to Sepharose (Sigma
Chemical Company, Poole, UK).
Immunofluorescence Microscopy--
20-day-old mice were perfused
intracardially with a 4% solution of paraformaldehyde in 0.1 M sodium phosphate, pH 7.4. Sciatic nerves were then
removed and fixed for a further 2 h at room temperature. After
washing, nerves were cryoprotected by immersion for 15 min in 5% (w/v)
and then 10% (w/v) sucrose in 0.1 M phosphate, pH 7.4, followed by overnight incubation in a 20% (w/v) solution at 4 °C.
Cryoprotected nerves were subsequently frozen in OCT embedding compound
(Tissue TEK) using isopentane. Transverse sections (7 µm) were
collected on 3-aminopropyltriethoxysilane-subbed glass slides.
Following removal of OCT by washing in phosphate-buffered saline (PBS)
(Sigma), sections were blocked for 3 h at room temperature in a
solution of 10% (v/v) goat serum (Scottish Antibody Production Unit,
Law Hospital, Carluke, Scotland), 0.2% (w/v) gelatin, and 0.3% (v/v)
Triton X-100 in PBS. Blocked sections were incubated overnight with
rabbit anti-170pep1 (1) (diluted 1:3000) or affinity-purified
anti-SPeri (diluted 1:200) with mouse anti-myelin basic protein
(diluted 1:200) (from Prof. N. Groome, Department of Biology, Oxford
Brookes University) in 4% (v/v) goat serum, 0.2% (w/v) gelatin and
0.3% (v/v) Triton X-100 (in PBS) under humid conditions at room
temperature. Slides were washed in blocking buffer minus goat serum and
then incubated with the secondary antibodies fluorescein
isothiocyanate-labeled goat anti-rabbit (Cappel, Durham, NC) (diluted
1:200) and biotinylated goat anti-mouse (Kirkegaard and Perry) (diluted
1:500). Both antibodies were in 4% (v/v) goat serum, 0.2% (w/v)
gelatin, and 0.3% (v/v) Triton X-100 (in PBS) and incubated for 1 h at room temperature. Further washes in blocking buffer minus serum
were followed by a 1-h incubation with streptavidin-Texas red (Vector
laboratories) (diluted 1:1500) and several washes in PBS. Sections were
subsequently examined with a Leica TCS4D confocal microscope.
Western Blot Analysis--
Sciatic nerve homogenates (10 µg)
were resolved by SDS-polyacrylamide gel electrophoresis on 4-20%
gradient polyacrylamide gels, and Western blotting was performed as
described previously (1). Rabbit anti-170pep1 (1) was diluted 1:2000,
affinity-purified rabbit anti-SPeri was diluted 1:200, and rabbit
anti-NTerm was diluted 1:1000. Goat anti-rabbit IgG-horseradish
peroxidase conjugate (Scottish antibody production unit) was used at a
dilution of 1:500. The peroxidase reaction was stopped by the addition
of SDS to 2% (v/v).
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RESULTS |
Intron-Exon Structure of Murine Periaxin Gene--
Three different
clones were isolated from a murine 129SV genomic library using a rat
periaxin cDNA as a hybridization probe. A fourth, which included
the 5'-end of the gene, was isolated using a murine cDNA probe that
had been generated by reverse transcription-PCR. The clones were
digested with EcoRI and subcloned into pIBI30. These clones
were analyzed by PCR and Southern blot and were sequenced, which
demonstrated that they encompassed the entire periaxin gene. The gene
is divided into seven exons, and the coding region spans were
approximately 20.6 kb (Fig. 1). Exons
1-6 range in size from 32 (exon 1) to 197 bp (exon 6). Exon 7 is the
largest by far at 4002 bp. The sizes of the introns were estimated by
restriction mapping and PCR and range from 88 (intron 2) to 7500 bp
(intron 5). The exon-intron boundaries were sequenced and the splice
donor (GT) and acceptor (AG) sites were identified. This information is
summarized in Table I.

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Fig. 1.
Structure of the murine periaxin gene.
Exons are numbered and indicated by solid rectangles.
Introns are shown as solid lines between the exons, except
for intron 6, which is depicted as a hatched box between
exons VI and VII. EcoRI (E) restriction sites are
indicated and were used to subclone the genomic clones F2, H1, and I1
(from the first screen) and HH1 (from the second screen).
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Table I
Exon/intron position, size, and junction sequence structure of the
mouse periaxin gene
The intronic 5'-splice donor GT and 3'-splice acceptor AG are in bold
type. The exonic sequences are capitalized. The interruption of codons
by introns is indicated by the phase. A phase of 0 indicates no
interruption, and insertion of an intron after the first nucleotide of
the codon is indicated by phase 1. All numbering is relative to the
deduced cDNA sequence of the mouse 4.6-kb periaxin message.
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Two distinct periaxin mRNAs are expressed in the rat peripheral
nervous system (1), and the murine gene also encodes two mRNAs of
4.6 and 5.2 kb of approximately equal abundance (Fig. 2A). Of eleven cDNA clones
isolated from a rat cDNA library, five included intron 6 (Fig. 1).
Confirmation that the murine 5.2-kb mRNA differed from the 4.6-kb
mRNA by the inclusion of this 592-bp intron was obtained by
Northern blotting (Fig. 2).

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Fig. 2.
Alternative splicing of a retained intron
generates two mRNAs. Sciatic nerve RNA from 15-day-old mice
was electrophoresed on a 0.8% agarose formaldehyde gel (2 µg/lane),
transferred to nylon membrane and probed in lane A with a
1.3-kb restriction fragment of nucleotides 1-1324 of the rat periaxin
cDNA and in lane B with a PCR product comprising intron
6 of the mouse gene. Both probes were labeled with
[ -32P]dCTP by random priming as described under
"Experimental Procedures." Autoradiography was for 48 h.
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Identification of Transcriptional Initiation Site and Core
Promoter--
The transcriptional initiation site was determined by
primer extension and was found to be 75 bp upstream of a primer
complementary to a region comprising the final nucleotide of exon 2 and
the 5'-end of exon 3 in the murine gene (Fig.
3). This site lay in the sequence
YYA+1AGGA, which has some similarity to the sequence
YYA+1N(A/T)YY believed to be the consensus transcriptional initiator for RNA polymerase II transcripts (16). An identical transcriptional initiation site was found for the rat mRNA (data not shown). Approximately 500 bp of the putative core promoter was
sequenced (Fig. 4). Although it lacks a
TATA box, the promoter does possess a CAAT box, and between this motif
and the initiator the sequence is relatively GC-rich (68%), which is
commonly the case in TATA-less promoters (17). A sequence motif
corresponding to the SCIP/Oct-6 binding site (position
241) is of
particular interest owing to the role that Oct-6 is believed to play in
Schwann cell maturation (18-20). An element (GCRE) at position
360
has previously been identified as mediating the induction of several myelin protein genes by forskolin (21). The presence of this sequence
would help to explain the ability of cAMP to mimic some of the axonal
signals that regulate the expression of differentiation-specific genes
in Schwann cells (22, 23).

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Fig. 3.
Determination of the periaxin transcription
initiation site by primer extension. An oligonucleotide
complementary to nucleotides 50-71 of rat periaxin cDNA (1) was
end-labeled with [ -32P]ATP and T4 polynucleotide
kinase. Total RNA (10 µg) from the trigeminal nerves of 13-day-old
mice was hybridized to labeled oligonucleotide (5 pmol) and reverse
transcribed. Extended products were resolved on a 6% polyacrylamide
sequencing gel adjacent to a sequencing ladder comprising the 5'-end of
periaxin primed using an oligonucleotide complementary to nucleotides
103-122 of mouse periaxin cDNA. (lane P).
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Fig. 4.
Nucleotide sequence of the transcriptional
initiation site and consensus sequences of transcription factor-binding
elements in the putative core promoter. Exon 1, which comprises 32 (positive numbers) and 491 bp of the putative core promoter
(negative numbers) are shown relative to the transcriptional
intiation site (+1) determined by primer extension. The
sequences recognized by STAT proteins (29), SCIP/Oct-6 (18-20), SREBP1
(30), and NF-Y (31) together with the consensus glial cAMP response
element (GCRE) (21) are underlined. The CAAT box
is double-underlined.
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Deduced Amino Acid Sequence of L-Periaxin and S-Periaxin--
The
deduced amino acid sequence of the protein encoded by the 4.6-kb
mRNA is depicted in Fig.
5A. This isoform, termed
L-periaxin, is 93% identical to rat periaxin and has a size of 147.500 kDa, slightly larger than the rat protein (1). The presence of a retained intron in the larger 5.2-kb mRNA introduces a stop codon preceded by a sequence that encodes a unique 21-amino acid C terminus. This truncated isoform, termed S-periaxin, has a size of 16.2 kDa (Fig.
5B). Eccept for two differences at the extreme C terminus, the rat and murine S-periaxins are identical. Anti-peptide antibodies recognizing the N terminus of L- and S-periaxin (anti-NTerm), the
repeat region unique to L-periaxin (anti-170pep1 (1)), or the C
terminus unique to S-periaxin (anti-SPeri) confirmed the structural
relationship between the two isoforms (Fig.
6).

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Fig. 5.
Comparison of the amino acid sequences of
mouse and rat periaxin. Alignment of the deduced amino acid
sequence of mouse and rat L-periaxin (A) and S-periaxin
(B) using the single-letter code. Identity is depicted by an
asterisk, and dashes indicate gaps introduced for
optimal alignment. The point at which L- and S-periaxin diverge in
sequence is shown by the arrows in A and B.
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Fig. 6.
Western blot of mouse periaxin with
domain-specific antibodies. Mouse sciatic nerves from 15-day-old
mice were homogenized and immunoblotted using antibodies raised against
peptide sequences comprising either the N terminus of L- and S-periaxin
(anti-NTerm), the repeat region unique to L-periaxin (anti-170pep1 (1))
or the C terminus unique to S-periaxin (anti-SPeri). The sizes of L-
and S-periaxin are indicated in kDa.
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A PDZ Domain at the N Terminus of L- and S-Periaxin--
The
similarity between mouse and rat periaxin is particularly striking in
the N-terminal 127 amino acids up to the point where the sequences of
L- and S-periaxin diverge. Within this region the rat and mouse
polypeptides are identical. This prompted us to examine this sequence
very carefully for conserved motifs that might illuminate the function
of these proteins, despite the fact that previous searches of the
complete L-periaxin polypeptide had not been informative (1). A degree
of sequence similarity with a portion of the PDZ domain of the
junction-associated protein ZO-1 provided the necessary clue.
Comparison of the N terminus sequence from amino acids 13 to 97 with
several well characterized PDZ domains in other proteins confirmed that
this region comprises a PDZ domain common to both L- and S-periaxin
(Fig. 7).

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Fig. 7.
Identification of a PDZ domain at the N
terminus of L- and S-periaxin. The sequence of mouse L- and
S-periaxin between amino acids 14 and 98 was compared with PDZ domains
in the Caenorhabditis elegans protein CET19B10 (residues
232-316) (32), PSD-95 (residues 309-393) (33), Discs-Large (dlg)
(residues 482-566) (34), SAP97 (residues 461-545) (35), ZO-1
(residues 408-491) (36), ZO-2 (residues 93-176) (37), and inaD
(residues 361-447) (38). The eight segments within the domain
comprising six strands and two -helices are as determined and
described by Doyle et al. (5). The sequences have been
arranged to maximize their similarity. Solid blocks show
amino acid identity, and shading indicates conservative
substitutions.
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Because proteins that contain PDZ domains are believed to associate
with the plasma membrane (3, 6), it was of considerable interest to
compare the subcellular locations of L- and S-periaxin in the Schwann
cell. In transverse sections of sciatic nerves from 20-day-old mice
L-periaxin was detected in a typical annular pattern that reflected its
concentration in the periaxonal and abaxonal myelin lamellae (Fig.
8, A and C). In
contrast to myelin basic protein, L-periaxin was also present at the
plasma membrane of the Schwann cell, consistent with the possession of
a PDZ domain. As found before, L-periaxin is not present in compact
myelin where myelin basic protein is abundant (Fig. 8, B and
C) (1, 2). In contrast, S-periaxin was not concentrated at
the interface between the plasma membrane and cytoplasm of the Schwann
cell or the myelin sheath (Fig. 8, D-F). Instead, this
protein seemed to be distributed fairly evenly throughout the
cytoplasm. There also appeared to be some S-periaxin in the nucleus of
the Schwann cell. Apparently, the presence of a PDZ domain may not be
sufficient to direct the association of S-periaxin with either the
Schwann cell plasma membrane or its product, the myelin sheath.

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Fig. 8.
Immunofluorescence localization of L- and
S-periaxin in mouse Schwann cells. A-C, transverse section
of a sciatic nerve from a 20-day-old mouse, double-labeled with
anti-170pep1 to detect L-periaxin (fluorescein isothiocyanate,
A) and antibodies against myelin basic protein
(MBP) (tetramethyl rhodamine isothiocyanate, B).
The combined image is shown in C. L-periaxin is localized to
the plasma membrane (arrow) and the abaxonal and adaxonal
surfaces (arrowheads) of myelinating Schwann cells. Myelin
basic protein is restricted to the myelin sheath. D-F,
transverse section of the same sciatic nerve double labeled with
anti-SPeri to detect S-periaxin (fluorescein isothiocyanate,
D) and anti-myelin basic protein (tetramethyl rhodamine
isothiocyanate, E). The combined image is shown in
F. S-periaxin is restricted to the cytoplasm of myelinating
Schwann cells. Preincubation of the anti-SPeri with 1 mg/ml of peptide
completely abolished staining with this antibody (data not
shown).
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DISCUSSION |
One of the most interesting aspects of the expression of the
periaxin gene is the fact that alternative splicing involves the
retention of an intron. There are approximately equal proportions of
two mRNAs that either lack or include the last intron. The factors
that determine the retention of introns have been studied in detail by
Rottman and colleagues, and a key feature is the "weakness" of the
splice sites at the 5'- and 3'-ends of the intron, i.e. the
extent to which they differ from the consensus sequences that are known
to promote splicing (12, 24). These weak splice sites would not
normally support splicing without an additional sequence in the
downstream exon called the exonic splicing enhancer (13). The main
distinguishing feature of exonic splicing enhancers is their high
content of purines (13). In support of this model, the splice sites in
the last intron of the periaxin gene are divergent from the consensus
sequences, and there is a domain downstream in exon 7 that is highly
purine-rich. The sequences of these 5'- and 3'-splice sites are
CTGgtacgc and tcagA, respectively (intron sequences in lowercase),
which are significantly different from the corresponding consensus
donor and acceptor sequences of CAGgtragt and ncagG (24). Downstream of
the acceptor site and 24 bases into exon 7 there is a 14-base sequence
GAAGAAGAAGAAGA, which is an excellent candidate for an exonic splicing
enhancer (1, 13).
Of those genes that are typically expressed by myelin-forming Schwann
cells, the periaxin gene is one of the first to become transcriptionally active (2). The means by which the gene is regulated
is therefore of considerable interest in evaluating how the expression
of those genes required for axon ensheathment and myelination is
coordinated. The SCIP/Oct-6 transcription factor is known to play a
part in Schwann cell maturation, and the presence of a binding site for
this protein in the periaxin promoter is intriguing (18-20). However,
the timing of expression of periaxin expression in SCIP/Oct-6 null
mutants appears to be relatively normal, despite the fact that
peripheral myelination is disrupted (20). The presence of a site that
bears considerable similarity to the consensus GCRE binding site found
in other genes that are characteristically expressed in myelin-forming
glia indicates that the the periaxin gene may respond to common
signaling mechanisms, in which cAMP can mimic to some extent the
inductive interactions of the axon (21). Certainly axonal contact has
been shown to be vital to maintain periaxin expression in Schwann cells
in vivo (2).
So far, two major functions have been ascribed to PDZ domains on the
basis of their interactions with plasma membrane proteins and their
presence in known signaling molecules such as dlg (6). First, they may act as organizers of cortical transduction complexes. In this model the PDZ protein acts as a scaffold on which the macromolecular signaling complex is assembled. These complexes have
been termed transducisomes, within which PDZ proteins are key elements
(25). Secondly, they may link transmembrane proteins with the actin
cytoskeleton via actin-binding proteins such as protein 4.1 (8, 9).
Indeed, the insolubilty of periaxin (actually L-periaxin) in Triton
X-100 was a key feature in the initial identification of this protein
as a potential cytoskeleton-associated protein (1, 3).
The discovery of a PDZ domain in the periaxin proteins is certain to
provide exciting new ways of interpreting the function not only of
periaxin but also of PDZ domain-containing proteins in general. The PDZ
motif in periaxin may either participate in the membrane-protein
interactions that are required to promote spiralization or act to
recruit proteins to a cortical scaffold important in transmembrane
signaling, at least for L-periaxin. The fact that S-periaxin is not
concentrated at the membrane-cytoplasm interface but is distributed
throughout the cytoplasm of the Schwann cell suggests that this protein
has a quite distinct function and that the PDZ domain may not be
sufficient to ensure cortical targeting. It is also possible that the
unique C terminus of S-periaxin might target the protein away from the
plasma membrane. Although most PDZ proteins are localized to the cell
cortex, there is an increasing recognition that they do not always
function at the plasma membrane (26). The possibility that they play a
part in nuclear signaling has been suggested by the fact that the tight junction-associated protein Z0-1 localizes to the nucleus before it is
recruited to sites of cell-cell contact (27). Furthermore, a nuclear
binding partner for the transcriptional factor SRY is a PDZ protein
that is thought to participate in a macromolecular transcription
complex (28).
In conclusion, we have shown that two periaxin isoforms, both of which
contain the same PDZ domain, are generated in approximately equal
amounts by an alternative retained intron mechanism. Furthermore, these
PDZ proteins are uniquely targeted to different localizations in
myelinating Schwann cells, which suggests that the presence of a PDZ
motif may not be the dominant determinant in the selection of binding
partners for L- and S-periaxin in myelinating Schwann cells.