From the Department of Neurology and
§ Paralyzed Veterans of America/Eastern Paralyzed Veterans
Association Neuroscience Research Center, Yale University School
of Medicine, New Haven, Connecticut 06510 and the ¶ Rehabilitation
Research Center, Veterans Affairs Medical Center,
West Haven, Connecticut 06516
Received for publication, February 20, 2001, and in revised form, March 15, 2001
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ABSTRACT |
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In this study we demonstrate a direct interaction
between a cytosolic fibroblast growth factor family member and a sodium channel. A yeast two-hybrid screen for proteins that associate with the
cytoplasmic domains of the tetrodotoxin-resistant sodium channel
rNav1.9a (NaN) led to the identification of
fibroblast growth factor homologous factor 1B (FHF1B), a member of the
fibroblast growth factor family, as an interacting partner of
rNav1.9a. FHF1B selectively interacts with the C-terminal
region but not the other four intracellular segments of
rNav1.9a. FHF1B binds directly to the C-terminal
polypeptide of rNav1.9a both in vitro and in mammalian cell lines. The N-terminal 5-77 amino acid residues of FHF1B
are essential for binding to rNav1.9a. FHF1B did not interact with C termini of two other sodium channels
hNav1.7a (hNaNE) and rNav1.8a (SNS), which
share 50% similarity to the C-terminal polypeptide of
rNav1.9a. FHF1B is the first growth factor found to bind
specifically to a sodium channel. Although the functional significance
of this interaction is not clear, FHF1B may affect the
rNav1.9a channel directly or by recruiting other proteins
to the channel complex. Alternatively, it is possible that
rNav1.9a may help deliver this factor to the cell membrane, where it exerts its function.
Ten distinct pore-forming The Peripheral sensory neurons in the dorsal root ganglia
(DRG)1 and trigeminal ganglia
produce pharmacologically and physiologically distinguishable
Na+ currents, some of which are sensitive to the
neurotoxin tetrodotoxin (TTX-S), and others which are resistant to this
toxin (TTX-R). The TTX-R Nav1.9a (NaN) sodium channel is
preferentially expressed in nociceptive neurons (14), and its
expression is modulated in response to axotomy (14) and inflammation
(15). The Nav1.9a channel produces a persistent TTX-R
Na+ current that activates at potentials of The yeast two-hybrid (Y2H) screening system (19) has proven to be an
effective tool in identifying cytosolic partners of ion channels
including voltage-gated K+ channels (20), AMPA
receptors (21), the Antibodies and Mammalian Cell Lines--
The preparation and
purification of an antiserum to rNav1.9 (NaN) has been previously
described (25). Anti-GFP and anti-Myc polyclonal antibodies were
purchased from CLONTECH. Human embryonic kidney
(HEK) 293 cells and mouse NIH3T3 fibroblasts were used.
Plasmids and Clones--
Yeast expression vector pDBleu (Life
Technologies) and pPC86 (Life Technologies) are fusion vectors for the
linkage of proteins to the Gal4 DNA binding domain and to the VP16
transactivation domain, respectively. The segment encoding the C
terminus (aa 1588-1765; GenBankTM accession number
AF059030) of rNav1.9a (NaN) was amplified by PCR and cloned
in-frame into the SalI/NotI sites of pDBleu (pDB-NaNC) to serve as bait in the screening assay. Fragments encoding
sub-regions of the C terminus of rNav1.9a were also
amplified and subcloned into the SalI/NotI sites
of pDBleu: CR1+CR2 (aa 1588-1727; pDB-NaNCR1CR2), CR1 (aa 1588-1658;
pDB-NaNCR1), CR2 (aa 1658-1727; pDB-NaNCR2), and UR (aa 1727-1765;
pDB-NaNUR). Fragments encoding other cytoplasmic components of this
channel were also cloned into the pDBleu vector: N terminus (aa 1-124; pDB-NaNN), L1 (aa 398-564; pDB-NaNL1), L2 (aa 802-1029; pDB-NaNL2), and L3 (aa 1288-1340; pDB-NaNL3). Similarly, cDNA fragments
encoding the C termini of rNav1.8 (SNS) (aa 1725-1957;
GenBankTM accession number X92184) and hNav1.7
(hNaNE) (aa 1753-1977; GenBankTM accession number X82835)
sodium channel were cloned in-frame into the
SalI/NotI sites of pDBleu vector to produce
plasmids pDB-SNSC and pDB-hNEC, respectively. The full coding sequence of rat FHF1B was cloned in-frame into the
SalI/NotI sites of pPC86 vector to generate
plasmid pPC86-rFHF1B.
The bacterial expression vector pGEX-3X (Life Technologies) was used to
produce recombinant proteins in Escherichia coli. The
respective C-terminal fragments of rNav1.9 described above were subcloned in-frame into the BamHI/EcoRI
sites of pGEX-3X to produce the plasmids pGEX-NaNC, pGEX-NaNCR1CR2,
pGEX-NaNCR1, pGEX-NaNCR2, and pGEX-NaNUR.
Mammalian two-hybrid expression vectors pBIND and pACT (Promega)
are fusion vectors for the linkage of proteins to the Gal4 DNA binding
domain and to the VP16 transactivation domain, respectively. The
complete coding sequence of rat FHF-1B cDNA was inserted in-frame into the SalI/NotI sites of pBIND fusion vector
to produce plasmid pBIND-FHF1B. A cDNA fragment encoding the C
terminus of rNav1.9a was inserted in-frame into the
SalI/NotI sites of pACT fusion vector to produce
plasmid pACT-NaNC. Plasmids for expression of tagged proteins in HEK293
cells were also constructed in the vectors pcDNA3.1/Myc-His (+)
(Invitrogen) and pEGFP-N1 (CLONTECH). A cDNA fragment encoding the C terminus of rNav1.9a (described
above) was inserted in-frame into the BamHI/EcoRI
sites of pcDNA3.1/Myc-His(+)A to produce plasmid
pcDNA3.1/Myc-His/NaNC. The complete coding sequence of FHF1B was
subcloned in-frame into the BamHI/EcoRI sites of
pEGFP-N1 vector (CLONTECH) to produce plasmid
pFHF1B-GFP. This construct produces FHF1B-GFP fusion protein when
introduced into mammalian cells.
All constructs were verified by sequencing their inserts. Sequencing
was carried out at the Howard Hughes Medical Institute/Keck Biotechnology Resource Laboratory at Yale University. Sequence analysis
was done using Lasergene (DNASTAR), GCG (GCG), and BLAST (National Library of Medicine) software.
RNA Preparation and Reverse Transcription-PCR--
Total
cellular RNA was isolated from tissues of adult Harlan Sprague-Dawley
rats by the single-step guanidinium isothiocyanate-acid phenol
procedure (26). First-strand cDNA was synthesized from total RNA as
previously described (27). PCR was carried out in two stages (27) for a
total of 27 cycles by using a PTC 200 programmable thermal cycler (MJ Research).
FHF1B and GAPDH primers were used in a multiplex PCR reaction to
determine the tissue distribution of FHF1B in adult rat tissues. FHF1B
forward (5'-CACTCTAAAGGGACCGAAATGGAG-3') and reverse
(5'-GAAGAGTTCTCAGCCAGGCTATG-3') primers amplify nucleotides 39-620
(GenBankTM accession number AF348446), whereas GAPDH
forward (5'-TCACCACCATGGAGAAGGCTG-3') and reverse
(3'-CCCTGTTGCTGTAGCCATATTC-3') primer amplify nucleotides 328-994
(GenBankTM accession number M17701). FHF1B and GAPDH
primers were used at final concentrations of 2 and 0.2 µM, respectively, to reduce the inhibition of FHF1B
amplification by excess GAPDH templates. Control PCR with water or RNA
template produced no amplification products (data not shown).
Yeast Two-hybrid (Y2H) Library Screen--
Plasmid pDBNaNC (see
above) was used as bait to screen rat brain pPC86 cDNA library
(Life Technologies) using the protocol provided by the manufacturer
with few modifications. Briefly, bait plasmid was first introduced into
yeast MAV203 strain that contains three reporter genes,
HIS+, URA+, and Lac Z (Life Technologies), and
transformants were selected on defined medium lacking leucine. The rat
brain cDNA library in pPC86 was then transformed into the resultant
Leu+ yeast strain and plated on medium lacking tryptophan,
leucine, histidine, and uracil but containing 25 mM
3-amino-1,2,4-trizone (SD
leu Assay of Protein-Protein Interactions by Y2H Assay--
Three
independent colonies were analyzed for the interaction in yeast between
two proteins, one of which was fused to the Gal4 DNA binding domain and
the other to the VP16 transactivation domain. The procedures of Vojtek
et al. (29) and Hollenberg et al. (28) were
followed for (i) growing and transforming the yeast strain MAV203 with
the selected plasmids and (ii) testing by the Expression and Purification of Glutathione S-Transferase (GST)
Fusion Proteins--
For expressing GST fusion proteins, the
appropriate plasmids (pGEX-NaNN (N terminus, aa 1-124); pGEX-NaNL1
(L1, aa 398-564); pGEX-NaNL2 (L2, aa 802-1029); pGEX-NaNC (C
terminus, aa 1588-1765), pGEX-NaNCR1CR2, pGEX-NaNCR1, pGEX-NaNCR2, and
pGEX-NaNUR (see above for coordinates)) were transformed into E. coli DH5 In Vitro Binding Assay--
To examine the binding of FHF1B and
its derivatives to the C terminus of rNav1.9 in
vitro, glutathione-Sepharose beads (50 µl) preincubated with
purified GST (0.5 µg), serving as control, or GST-NaNC (0.5 µg)
were incubated with extract (500 µg of protein) of HEK293 cells
transfected with an expression plasmid encoding either full-length
FHF1B-GFP or its derivatives (i.e. FHF1B-(5-181)-GFP, FHF1B-(42-181)-GFP, FHF1B-(77-181)-GFP, and FHF1B-(143-181)-GFP), as
indicated, in 150 µl of buffer AM (10 mM Tris, pH. 7.9, 10% glycerol, 1 mM MgCl2) (31) supplemented
with 100 mM KCl and 0.5 mg/ml bovine serum albumin (30).
The bound proteins were denatured in sample buffer and separated by
12% SDS-PAGE, and GFP fusion proteins were detected by immunoblotting
with anti-GFP antibodies (CLONTECH). Similarly, GST
fusion proteins of C terminus derivatives (0.5 µg) were analyzed in
this binding assay to further delineate the sequence in the C terminus
of rNav1.9 that is responsible for the interaction with FHF1B.
Far Western Assay--
Far Western (overlay blot) assay was
carried out according to Lee et al. (32) with minor
modification. After a 1-h incubation with anti-GFP antibodies, extracts
of HEK293 cells transfected with an expression plasmid encoding either
GFP or FHF1B-GFP fusion protein were incubated with protein A-agarose
at 4 °C overnight. After washing five times in cell lysis buffer
(phosphate-buffered saline, pH 7.5, 1% Triton X-100, 5 mM
EDTA), the purified GFP (control), and FHF1B-GFP were loaded onto 15%
SDS-PAGE and subjected to electrophoresis. The proteins were
electrotransferred to a nitrocellulose membrane at 85 mA for 2 h.
The proteins on the blot were denatured and renatured by sequential
washings in 0.1 M CZ solution (20 mM HEPES, pH
7.9, 20% glycerol, 0.1 M KCl, 5 mM
MgCl2, 01.mM ZnCl2, 0.1 mM EDTA, 2 mM dithiothreitol) plus 0.02%
polyvinylpyrrolidone and 6 M guanidine-HCl for 30 min, one time, then three times in 0.1 M CZ solution plus 0.02%
polyvinylpyrrolidone, each for 2 h. After blocking with 5% bovine
serum albumin, the membranes were incubated with 50 µg of purified
GST-NaNC protein, followed by incubation with anti-NaN antibodies (0.2 µg/ml) for 1 h. The signal was detected using the ECL
chemiluminescent system (Amersham Pharmacia Biotech).
Two-hybrid Assay for Interacting Proteins in Mammalian
Cells--
Components of the mammalian two-hybrid assay (Promega) were
used to study the interaction of FHF1B and the C terminus of
rNav1.9 in NIH3T3 cells. NIH3T3 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 100 unit/ml antibiotics. When cultures reached about
50% confluence, the cells were transfected with 1 µg each of the
indicated plasmids using Lipofectin (Life Technologies) according to
the manufacturer's recommendations. Plasmid pUC19 was used as a
carrier to bring the total amount of DNA in the transfection solution
to 3 µg. The cultures were harvested 48 h after transfection and
lysed, and firefly luciferase activity was determined according to
manufacturer's recommendations.
Co-immunoprecipitation--
HEK293 cells were grown in 100-mm
dishes in Dulbecco's modified Eagle's medium containing
heat-inactivated 10% fetal bovine serum. After reaching 50%
confluence, the cultures were co-transfected with the mammalian
expression plasmids pFHF1B-GFP and pcDNA3.1Myc-His/NaNC. The
cultures were harvested 48 h later, and cell extracts were prepared according to Liu et al. (33). After a 1-h
incubation with either anti-Myc (25 µg/ml)
(CLONTECH) or anti-NaN (20 µg/ml) antibodies,
aliquots (200 µl) from this mixture were incubated with 30 µl of
protein A-agarose (Life Technologies) at 4 °C overnight. After
washing five times with immunoprecipitation buffer (34), the bound
proteins were released by boiling in 20 µl of 2× SDS loading buffer
for 3 min (30). The released proteins were examined by Western blotting
with anti-GFP antibodies (CLONTECH), and the signal
was detected using the ECL chemiluminescent system (Amersham Pharmacia Biotech).
Identification of FHF1B as a Binding Partner of
rNav1.9a--
The tetrodotoxin-resistant (TTX-R) sodium
channel rNav1.9a (NaN) is expressed preferentially in
peripheral sensory neurons and is down-regulated after axotomy (14).
This channel is also expressed at low levels in central nervous system
tissues (14, 35). A screen based on the yeast two-hybrid (Y2H) system
(19) was performed to identify proteins that interact with
rNav1.9a. For this purpose, we linked the five
intracellular regions of rNav1.9a, namely, the N terminus,
loop 1, loop 2, loop 3, and the C terminus to the Gal4 DNA binding
domain (GAL4DBD) in the plasmid pDBleu. We used the respective
constructs as baits to screen a library of rat brain cDNA expressed
as fusion proteins to the VP16 acidic activation domain (VP16AD) in
the vector pPC86.
A Y2H rat brain cDNA library (Life Technologies) was screened with
the construct encoding the cytosolic C terminus of rNav1.9a (aa 1588-1765). We screened about 5 million clones and identified 23 clones that activated the three reporter genes. Further tests involved
the re-transformation of yeast with the purified target plasmids and
bait. Only 2 of the original 23 yeast clones expressed full-length
proteins that interact with the C-terminal bait (not shown). The
plasmids in these clones contained 2.96-kilobase pair identical inserts.
Sequence Analysis of Full-length rFHF1B--
The sequence of the
two inserts matched that of the chicken fibroblast growth factor
homologous factor gFHF1B (36) and represents the rat homologue of this
factor (Fig. 1). This factor belongs to
the FGF family of growth factors (24). An open reading frame of 546 base pairs encodes a polypeptide of 181 amino acids with a predicted
mass of 20 kDa (Fig. 1A). The rat factor, rFHF1B, is highly
conserved compared with the chicken counterpart with 87% identity at
the nucleotide level (not shown) and 97% identity at the amino acid
level (Fig. 1B). FHF1B lacks a canonical signal peptide, like the other members of this subfamily (24). Unlike FHF1A,
which possesses a nuclear targeting signal as a result of alternative
splicing, rFHF1B lacks such a signal (Fig. 1B).
Multiplex PCR of FHF1B and GAPDH was used to investigate tissue
distribution of FHF1B in adult rat tissue. FHF1B is readily detectable
in central and peripheral nervous tissues including cerebrum,
cerebellum, spinal cord, DRG, and trigeminal ganglia (Fig.
2). The rFHF1B fragment that is amplified
from DRG tissues using these rFHF1B-specific primers shows identical
sequence to that obtained from the rat brain cDNA library. This
factor is expressed, however, at lower levels in other neural tissues
such as superior cervical ganglia and retina and in the cardiac and skeletal muscles, whereas it is undetectable under these amplification conditions in liver, and kidney (Fig. 2). The tissue distribution of
rFHF1B shows an overlap with the expression of rNav1.9a in DRG and
trigeminal ganglia (14).
The Y2H assay was repeated to verify the interaction between rFHF1B and
the C terminus of rNav1.9a. For this purpose, the plasmid
encoding the C terminus of rNav1.9a linked to the Gal4DBD and the plasmid encoding rFHF1B fused to the VP16AD were used to
co-transform the yeast strain MAV203. Plasmid pairs encoding c-Jun/c-Fos and Rb/lamin were used as positive and negative
protein-protein interaction controls, respectively (Fig.
3A). The interaction between
the C terminus polypeptide of rNav1.9a and rFHF1B was verified by
The other cytoplasmic polypeptides of rNav1.9a (Fig.
3B) were tested for their ability to interact with rFHF1B.
Filter-based rFHF-1B Binds Directly to the C-terminal Polypeptide of
rNav1.9a in Vitro--
To test whether rFHF1B binds to the
C-terminal polypeptide of rNav1.9a in vitro, we
expressed rFHF1B-GFP fusion protein in HEK293 cells and expressed the
C-terminal polypeptide of rNav1.9a as a GST fusion protein
(GST-NaNC). Affinity-purified GST and GST-NaNC immobilized on
glutathione-Sepharose beads were incubated with cell lysates expressing
either GFP or FHF1B-GFP. Purified GST or GST-NaNC did not pull down GFP
protein in this assay (Fig. 4A, lanes 1-3).
Purified GST did not pull down rFHF1B-GFP fusion protein (Fig.
4A, lane 5), whereas GST-NaNC efficiently pulled down the rFHF1B-GFP fusion protein (Fig. 4A, lane
6).
We used an overlay binding assay to show that rFHF1B binds directly to
the C-terminal polypeptide of rNav1.9a. GFP or rFHF1B-GFP proteins were immunoprecipitated with anti-GFP antibodies from lysates
of HEK293 cells transfected with the corresponding expression plasmid.
These immunoprecipitated proteins were separated by SDS-PAGE electrophoresis, electrotransferred and immobilized onto nitrocellulose membrane, and incubated with affinity-purified GST or GST-NaNC. Binding
was then detected using anti-NaN antibodies. Fig. 4B shows that the purified GST-NaNC fusion protein binds to the purified rFHF1B-GFP (lane 2) but not to GFP alone (lane
1); GST did not bind to either rFHF1B-GFP or GFP (not shown).
FHF-1B Binds to the C-terminal Polypeptide of rNav1.9a
in Vivo--
To test whether rFHF1B binds to the C-terminal
polypeptide of rNav1.9a in vivo, we performed a
mammalian two-hybrid assay in NIH3T3 cells containing a reporter
plasmid in which the expression of the firefly luciferase gene was
driven by five Gal4-specific enhancer elements (Fig.
5A). By analogy to the Y2H
system, the interaction between the C-terminal polypeptide of
rNav1.9a and rFHF1B brings together the Gal4DBD and the
VP16AD of the fusion proteins and activates the reporter gene in NIH3T3
cells (Fig. 5A). Fig. 5B shows that these two
proteins interact in mammalian cells as well as in yeast. Therefore,
any post-translational modifications that may occur in mammalian cells,
but that are absent in yeast, do not interfere with the interaction of
rFHF1B and the C-terminal polypeptide of rNav1.9a.
The result of the mammalian two-hybrid assay was confirmed by a
co-immunoprecipitation assay (Fig. 5C). In this assay, the extracts of cells expressing both rFHF1B-GFP fusion protein (lane 1) and Myc-tagged C-terminal polypeptide of rNav1.9a
were first incubated with either anti-Myc or anti-NaN antibodies, and
the immunoprecipitated complexes were counter-tested with anti-GFP antibodies. A clear rFHF1B-specific band was present in the
immunoprecipitated complexes brought down by either anti-Myc
(lane 2) or anti-NaN (lane 3) antibodies,
demonstrating that rFHF1B binds to the C terminus polypeptide of
rNav1.9a in vivo.
The N-terminal Segment (aa 5-77) of rFHF1B Is Required for
Association with the C-terminal Polypeptide of
rNav1.9a--
A series of N-terminal deletions of rFHF1B
were linked to GFP (Fig. 6A).
Pull-down assays involving the immobilized GST (Fig. 6B,
lanes 2, 5, 8, 11, and
14) and GST-NaNC (Fig. 6B, lanes 3, 6, 9, 12, and 15) and cell
extracts prepared from HEK293 cells transfected with rFHF1B mutants
fused to GFP (Fig. 6B, lanes 1, 4,
7, 10, 13) were performed to determine
the ability of the various rFHF1B mutants to associate with the
C-terminal polypeptide of rNav1.9a. Immunoblotting using
anti-GFP antibodies shows that rFHF1B lacking the N-terminal
pentapeptide binds GST-NaNC as efficiently as the full-length factor
(compare lanes 3 and 6). Deletion of the
N-terminal 42 residues significantly reduced the interaction of the two
polypeptides (lane 9), whereas removal of 77 residues totally disrupted this interaction (lane 12). Not
surprisingly, rFHF1B lacking the N-terminal 143 residues does not show
any interaction with GST-NaNC (lane 15). As a negative
control, GST did not pull down either of the rFHF1B derivative
polypeptides (Fig. 6B, lanes 2, 5,
8, 11, 14). These data show that the
N-terminal segment (aa 5-77) contains the molecular determinants for
the interaction of rFHF1B with the C-terminal polypeptide of
rNav1.9a.
Conserved Segments of the C-terminal Polypeptide of
rNav1.9a Are Important for rFHF1B Binding--
Alignment
of the C-terminal polypeptides of all mammalian rFHF1B Binds Specifically to rNav1.9a--
The length
of the C-terminal polypeptides of the Na+ channel
We used the yeast two-hybrid (Y2H) screen to identify protein
partners of the TTX-R sodium channel rNav1.9a (NaN). We
present evidence that rFHF1B, which is a member of the FGF family of
growth factors, binds directly to the C-terminal polypeptide of
rNav1.9a. This interaction is specific to
rNav1.9a and is not seen in other Na+ channels
that, like rNav1.9a, are preferentially expressed in DRG
and trigeminal neurons. We also present evidence that localizes the
binding site to specific segments of rNav1.9a C terminus
polypeptide and rFHF1B, respectively. Our results provide the first
evidence for a direct link between a growth factor and a voltage-gated sodium channel.
Sodium channels interact with multiple protein partners, affecting
their localization and the amplitude and kinetics of the currents that
they produce. The The TTX-R sodium channel rNav1.9a is preferentially
expressed in small diameter C-type sensory neurons of DRG and
trigeminal ganglia (14). The expression of rNav1.9a is
dynamic and is modulated by the action of neurotrophic factors. Rat
Nav1.9a transcripts and protein and the current
attributable to rNav1.9a are significantly down-regulated
in vivo after axotomy or chronic constriction injury of the
sciatic nerve (14, 17, 47) and in an in vitro model of
axotomy (47, 48). Expression of rNav1.9a after axotomy is
restored by exposure to the neurotrophic factor glial derived neurotrophic factor, but not nerve growth factor (47, 48).
The Nav1.9a channel has unique properties compared with
other sodium channels. The Nav1.9a channel is persistent at
This study presents evidence from multiple independent assays that
demonstrates the direct interaction of rFHF1B with the C-terminal
polypeptide of rNav1.9a both in vivo and
in vitro. Despite the 50% similarity of the amino acid
sequence of the C-terminal polypeptides of sodium channels, rFHF1B did
not bind the C termini of hNav1.7 (hNaNE) and
rNav1.8a (SNS), the two other sodium channels that are
preferentially expressed in DRG neurons. Although the conserved region
CR1 of rNav1.9a (aa 1588-1657) is sufficient to bind this
factor, the full conserved region CR1+CR2 (aa1588-1726) is needed for
efficient binding with rFHF1B. These data clearly demonstrate the
specific interaction between the C-terminal polypeptide of rNav1.9a and rFHF1B.
FHFs constitute a distinct branch of the FGF family with significant
homology to other FGF members in the central domain but with divergent
N and C termini (24). Alternative splicing produces variants of FHFs
that contain a nuclear localization signal and others that lack such
signals (24, 50-52). All FHF members discovered to date that lack a
nuclear localization signal (24, 50-52), like FGF-1 and FGF-2, also
lack a canonical signal peptide for secretion via the endoplasmic
reticulum/Golgi pathway. Consistent with the lack of signal peptides,
recombinant FHFs expressed in HEK293 cells are not secreted into the
culture media (24). Although some FHF isoforms can interact with
extracellular heparin sulfate proteoglycans, they do not activate FGF
receptors nor have they been directly linked to a biological activity
(36, 53). The ectopic expression of FHF-2 in chicken limb buds,
however, leads to morphological abnormalities, suggesting that this
factor play a role in pattern formation (36).
Like other members of the FGF family of growth factors, FHF1B is highly
conserved among various species. The similarity at the amino acid level
between full-length rat (this study) and chicken (36) FHF1B is 97%.
Rat FHF1B is 100 and 97% similar, respectively, to the corresponding
sequences that have been reported for mouse and human FHF1B (52). The
expression of rFHF1B in adult rat tissue as determined by reverse
transcription-PCR (Fig. 2) is similar to its expression pattern in the
chicken (36), except for the presence of rFHF1B in adult rat DRG and
trigeminal tissue. The expression of FHF1B, however, is more extensive
than that of rNav1.9a. Therefore, this factor may interact with other proteins, possibly including other sodium channels. More experiments are needed to determine if rFHF1B interacts with sodium channels that
are expressed in the brain.
The lack of a canonical signal peptide and nuclear localization signal
in rat and chicken FHF1B suggests that FHF1B may exert its effect via
interaction with cytosolic proteins either directly or by acting as a
bridge to assemble a functional protein complex. Alternatively, FHF1B
may be exported to the extracellular space via an endoplasmic
reticulum/Golgi-independent mechanism similar to that responsible for
FGF2 export (54), which is inhibited by cardenolides, suggesting a role
of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits of sodium channels have
been identified in the rat, and homologues have been cloned from various mammalian species including humans (1). Various
-subunits are expressed in a tissue- and developmentally specific manner (2).
Aberrant expression patterns or mutations of voltage-gated sodium
channel
-subunits underlie a number of human and animal disorders
(3-7), including neuropathic pain (8, 9).
-subunits of sodium channels have been shown to interact
with a number of proteins in addition to the auxiliary
subunits of
the channel that affect their subcellular localization, their amplitude, and/or their kinetic properties. The C terminus of skeletal
muscle and cardiac muscle
-subunits, SkM1 and SkM2, respectively,
bind to syntrophin via their PDZ domain, thus linking these channels to
the cortical actin network and extracellular matrix through the
association of syntrophin with the dystrophin-associated protein
complex (10). Brain
-subunits are phosphorylated at specific
serine/threonine sites by protein kinase A that is anchored to the
-subunit via AKAP15 (11, 12). Receptor protein-tyrosine phosphatase
(RPTP
) interacts with brain
-subunits via its carbonic anhydrase
homology extracellular domain and its intracellular phosphatase domain
and phosphorylates tyrosine residues of the channel (13).
60 to
70 mV
and is predicted to contribute to setting the resting membrane
potential and modulating subthreshold electrogenesis in these neurons
(16-18).
2 glutamate receptor (22), and voltage-gated
Na+ channels (23). To identify proteins that interact with
the rNav1.9a C terminus, we screened brain Y2H cDNA
library using a construct that encodes the C-terminal 198 residues of
rNav1.9a as bait. These experiments identified fibroblast
growth factor homologous factor 1B (FHF1B), which belongs to a subset
of the FGF family (24), as a binding partner of rNav1.9a.
It is possible that this interaction may help in exporting FHF1B to the
exterior face of the cell membrane, or it may modulate the properties
of rNav1.9a directly or indirectly by recruiting other
factors to the channel complex.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/trp
/his
/ura
/3AT+).
After incubation for 7 days at 30 °C, colonies were screened for
-galactosidase by a filter lift assay (28). Individual pPC86
recombinant plasmids were further verified for interaction with bait by
repeating the Y2H assay.
-galactosidase assay
and by growth phenotypes on (SD
leu
/trp
/his
/ura
/3AT+)
plates and on plates containing 5-fluoroorotic acid (SD
leu
/trp
/5FOA+).
(Life Technologies). The fusion proteins were
affinity-purified on glutathione-agarose beads as previously described
(30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence of rat FHF1B. A,
deduced amino acid sequence of rFHF1B. The sequence of rFHF1B was
deposited into GenBankTM (accession number AF348446). The
FGF family signature motif is in boldface type. Recognition
sites of multiple protein kinases were identified in this sequence:
protein kinase A site (RKSS, aa 162-165); PKC sites (SGR, aa 107-109;
SRK, aa 161-163); casein kinase 2 (TKDE, aa 34-37; SVFE, aa 88-91;
SLHE, aa 150-153). B, amino acid sequence alignment of
rFHF1B and other members of the FHF1 subfamily. Stars
represent residues that are identical to those of rFHF1B. The bipartite
components of the nuclear localization signal in FHF1A are underlined.
h, human; m, mouse; g, chicken; and
r, rat.
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Fig. 2.
Tissue distribution of rFHF1B by reverse
transcription-PCR. Amplification products from DRG, trigeminal
ganglia, cerebral hemisphere, cerebellum, and spinal cord (lanes
1-5, respectively) are consistent with a predicted size of 581 base pairs (bp) for rFHF1B and 652 base pairs for GAPDH. A
faint rFHF1B signal is detectable in superior cervical ganglia, retina,
cardiac muscle, and skeletal muscle (lanes 6-9). No
detectable rFHF1B signal is seen in liver (lane 10) and
kidney (lane 11). All samples show a GAPDH amplification
product, confirming that the absence of rFHF1B signal is due to lack of
gene expression rather than the quality of the cDNA template.
M lanes contain the 100-base pair ladder marker
(Amersham Pharmacia Biotech).
galactosidase assay and growth phenotype on selective media (Fig. 3A). Like the c-Jun/c-Fos pair, which is known
to interact, our assays indicate that rFHF1B interacts with the C terminus of rNav1.9a in yeast, based on the activation of
the LacZ reporter gene (Fig. 3A, left panel),
growth on the plates lacking histidine and uracil but containing
3-amino-1,2,4-trizole (Fig. 3A, middle panel),
and inhibition of growth on plates containing 0.2% 5-fluoroorotic acid
(Fig. 3A, right panel).
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Fig. 3.
Binding of rFHF1B to the C-terminal
polypeptide of rNav1.9a. A, yeast
two-hybrid assay to test the interaction between proteins fused to the
VP16 AD and proteins fused to the Gal4 DBD. Each pair of plasmids, as
indicated, encoding proteins fused to VP16 (above the line)
in the vector pPC86 (i.e. pPC86-c-jun, pPC86-FHF1B, and
pPC86-Rb) and those encoding proteins fused to Gal4 (under the
line) in the vector pDBleu (i.e. pDB-c-fos,
pDB-NaNC, and pDB-lamin) were co-transfected into yeast strain MAV203.
Yeast transformants were selected on SD
leu /trp
plates and tested for
-galactosidase activity (left panel) for growth on plates
lacking histidine, uracil, and containing 3AT (middle panel,
SD
leu
/trp
/his
/ura
/3AT+)
and for growth on plates containing 5 fluoroorotic acid
(5FOA) (right panel, SD
leu
/trp
/5FOA+).
The known interaction between c-Jun and c-Fos is used as a positive
control, whereas the lack of interaction of Rb and lamin serves as a
negative control. B, schematic diagram of the
rNav1.9a. N terminus (N-term, aa 1-124), loop 1 (L1, aa 398-564), loop 2 (L2, aa 802-1029), loop 3 (L3, aa
1288-1340), and the C terminus (C-term, aa 1588-1765)
represent the five cytoplasmic regions of the sodium channel. The four
domains comprising the
-subunit are designated I-IV. C,
comparison of the binding of rFHF1B to the individual intracellular
segments of rNav1.9a. A plasmid encoding the full coding
sequence of rFHF1B fused to the VP16AD in the vector pPC86 and a
plasmid encoding one of the five cytoplasmic region of
rNav1.9a fused to the Gal4DBD in the vector pDBleu were
co-transfected into the MAV203 yeast strain. Yeast transformants were
selected on SD leu
/trp
plates. Three
independent yeast transformants for each pair of plasmids were
transferred onto a nitrocellulose membrane, and the
-galactosidase
activity was determined as previously described (28, 29).
-galactosidase assays were used to determine if the
co-expression of the various rNav1.9a/Gal4DBD and
rFHF1B/VP16AD fusion proteins activated the reporter LacZ gene. As
shown in Fig. 3C, rFHF1B selectively interacts with the
C-terminal polypeptide of rNav1.9a, among the five
intracellular polypeptides tested in this assay.
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Fig. 4.
Direct interaction between rFHF1B and the C
terminus polypeptide of rNav1.9a in
vitro. A, binding of rFHF1B to NaNC in
vitro (GST pull-down assay). Purified GST (lanes 2 and
5) or GST-NaNC fusion protein (lanes 3 and
6) immobilized on glutathione-Sepharose beads were incubated
with extracts prepared from HEK293 cells transfected with a plasmid
encoding either GFP alone (lanes 1-3) or rFHF1B-GFP fusion
protein (lanes 4-6). HEK293 proteins that are trapped by
the interaction with GST or GST-NaNC (lanes 1 and
4, respectively) were examined by immunoblotting with
anti-GFP antibodies. GFP and rFHF1B-GFP as well as an unknown protein
are indicated. B, direct interaction of rFHF1B with NaNC as
determined by Far Western blotting. GFP (lane 1) or
rFHF1B-GFP (lane 2) proteins, immunoprecipitated by anti-GFP
antibodies from HEK293 cells transfected by the corresponding plasmid,
were separated by 12% SDS-PAGE, electroblotted onto nitrocellulose
membranes. The membranes were incubated with 50 µg of purified
GST-NaNC followed by immunodetection using anti-NaN antibodies.
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Fig. 5.
Interaction between rFHF1B and C terminus
polypeptide of rNav1.9a in vivo. A,
schematic representation of the two-hybrid assay in mammalian cells.
The expression of the firefly luciferase is driven by five copies of
the Gal4 DNA recognition sequence in the reporter plasmid (pG5luc). A
fragment encoding the full open reading frame of rFHF1B is linked to a
sequence encoding Gal4DBD in the vector pBIND-FHF1B. A fragment
encoding the C terminus of rNav1.9a is linked to a sequence
encoding the VP16AD in the vector pACT-NaNC. B, the
plasmids, as indicated, were transfected into NIH3T3 cells. After
incubation, the firefly luciferase activity was determined in cell
lysates. C, co-immunoprecipitation (Ip) assay of
fusion proteins expressed in HEK293 cells. Extracts prepared from
HEK293 cells co-transfected with two expression plasmids encoding
rFHF1B-GFP and Myc-tagged NaNC hybrid proteins were incubated with
either polyclonal Myc antibodies (lane 2) or NaN antibodies
(lane 3). The immunoprecipitated protein complex and cell
extract (lane 1, which provides a positive control) were
examined by immunoblotting (Wb) with an anti-GFP antibody.
Bands for FHF1B-GFP, IgG, and protein X are indicated.
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Fig. 6.
Identification of rFHF1B segments binding to
rNav1.9a. Schematic diagram of FHF1B-GFP constructs
used to map the binding of FHF1B to rNav1.9a.
Numbers refer to the amino acid residues in FHF1B. Each
segment (i.e. aa 1-4, 5-41, 42-76, 77-142, and 143-181)
is encoded by a single exon. The binding or lack of binding of the
various FHF1B segments to rNav1.9a, as revealed in
B, are indicated by plus or minus signs, respectively.
B, 0.5 µg of GST (lanes 2, 5,
8, 11, and 14) or 0.5 µg of GST-NaNC
(lanes 3, 6, 9, 12, and
15) immobilized on glutathione-Sepharose beads was incubated
with cell extracts prepared from HEK293 cells transfected with an
expression plasmid encoding either full-length FHF1B-(1-181)-GFP
(lanes 1-3), FHF1B-(5-181)-GFP (lanes 4-6),
FHF1B-(42-181)-GFP (lanes 7-9), FHF1B-(77-181)-GFP
(lane 10-12), or FHF1B-(143-181)-GFP (lane
13-15). The bound proteins and cell extracts (lanes 1,
4, 7, 10, and 13) were
examined by immunoblotting with anti-GFP antibodies. For additional
details, see "Experimental Procedures."
-subunits (not
shown) allowed us to divide this part of rNav1.9a polypeptide into two conserved regions (CR1 and CR2) of about 70 amino
acids each and a terminal unique region (UR) of 38 amino acids. We
subcloned fragments encoding the individual conserved and unique
regions to identify the molecular determinants of rFHF1B binding. We
first carried out Y2H assays using CR1, CR2, CR1+CR2, and UR GAL4DBD
baits and full-length rFHF1B fused to VP16AD. These experiments
demonstrate that conserved region 1 (CR1, aa 1588-1657) alone or
together with conserved region 2 (CR1+CR2, aa 1588-1726) of the
C-terminal polypeptide interact with rFHF1B, whereas the unique region
(UR, aa 1727-1765) did not interact with this factor (data not shown).
We also used a GST pull-down assay to determine quantitatively the
efficiency of the interaction of the various constructs with rFHF1B
(Fig. 7A). In these assays,
comparable amounts of the various purified GST fusion proteins (Fig.
7C) were preincubated with glutathione-Sepharose beads (50 µl) followed by incubation with cell extracts from HEK293 cells
expressing rFHF1B-GFP fusion protein (Fig. 7B, lane
1). The results confirm that the UR was neither capable of binding
to this factor (Fig. 7B, lane 7) nor did its
removal affect the interaction between rFHF1B and the conserved regions
of the polypeptide (Fig. 7B, lane 4). Like the
unique region, conserved region 2 alone did not interact with rFHF1B
(Fig. 7B, lane 6). The removal of CR2, however,
significantly reduced the strength of the interaction of CR1 with
rFHF1B (compare lanes 4 and 5 of Fig.
7B). These results suggest that the binding site of rFHF1B
straddles the CR1-CR2 junction or that CR2 influences the folding of
CR1 such that the molecular determinants of rFHF1B binding are
optimally accessible when CR2 is present. Further experiments are
needed to distinguish between these two possibilities.
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Fig. 7.
rFHF1B binds to the conserved region in the C
terminus of rNav1.9a. A, schematic diagrams
of GST-NaNC fusion proteins used to map the sites of rFHF1B binding on
the C-terminal polypeptide of rNav1.9a. The
numbers refer to amino acid residues in the C terminus of
rNav1.9a. CR1, CR2, and UR stand for conserved region 1, conserved region 2, and unique region, respectively. The strengths of
the binding of rFHF1B to the various GST-NaNC segments as revealed in
B are indicated. B, binding of rFHF1B by the C
terminus of rNav1.9a and its derivatives.
Glutathione-Sepharose beads carrying GST and GST-NaNC or its
derivatives as indicated were incubated with extracts prepared from
HEK293 cells transfected with an expression plasmid encoding rFHF1B-GFP
fusion protein, and the bound FHF1B-GFP was detected by immunoblotting
with anti-GFP antibodies. The FHF1B-GFP band is indicated.
C, expression of free GST or GST linked to the C-terminal
polypeptide of rNav1.9a or its derivatives. Samples of
affinity-purified GST and GST-NaNC or its derivatives (0.5 µg) as
indicated were examined by SDS-PAGE and Coomassie Blue staining. The
positions of size markers in kDa are indicated.
-subunits from rat tissues varies from 177 for rNav1.9a
to 251 for rNav1.3a (brain type III). Alignment of these
polypeptides (not shown) shows that the similarity of the C-terminal
polypeptide of rNav1.9a ranges from 50% for
rNav1.1a (brain type I) to 56.7% for rNav1.5a
(rSkM2). This similarity is 51.1 and 53.4% to rNav1.8a (SNS) and hNav1.7a (hNaNE), respectively, both of which,
like Nav1.9a, are expressed preferentially in DRG and
trigeminal neurons (37, 38). Therefore, we tested whether the
C-terminal polypeptides of these two channels are capable of binding
the rFHF1B protein in Y2H assays. The data in Fig.
8 shows that rFHF1B selectively binds to
the C-terminal polypeptide of rNav1.9a but not to those of
rNav1.8a or hNav1.7a.
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Fig. 8.
rFHF1B selectively associates with the
C-terminal of rNav1.9a. The -galactosidase assay
was used to test the interaction between rFHF1B and the C termini of
the sodium channels rNav1.9a (NaNC), hNav1.7a
(hNEC), and rNav1.8a (SNSC). The known interaction between
c-Jun and c-fos serves as a positive control, whereas the lack of
interaction of Rb and lamin serves as a negative control. Three
independent yeast transformants for each pair of plasmids were
transferred onto the nitrocellulose membrane, and the
-galactosidase
activity was determined as previously described (28, 29).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 subunit is proposed to increase the delivery of
pore-forming
-subunits to the cell membrane (39), whereas the
1
subunit modulates the amplitude and rate of inactivation of recombinant
-subunits expressed in Xenopus oocytes (40, 41) and
permits interaction of the sodium channel complex with the
extracellular matrix proteins tenascin-C and -R (42-45). Skeletal and
cardiac muscle sodium channels interact directly with the cytoskeletal
element syntrophin, which may determine their localization (10). The
interaction of brain sodium channels with AKAP15 is required for
dopaminergic modulation (11). More recently, the C-terminal polypeptide
of rNav1.2 (brain type II) was shown to bind calmodulin,
suggesting modulation of these channels by Ca2+ (23), and
the rNav1.2-L1 was shown to interact with synaptotagmin in
a Ca2+-regulated manner (46). An interaction with
synaptotagmin may modulate sodium channel activity by controlling the
accessibility of a protein kinase A phosphorylation site located inside
the binding region of this complex or may play a role in the
internalization of sodium channels (46). A more intriguing possibility
is that this interaction may result in
Na+-dependent exocytosis (46).
60 to
70 mV and shows a significant window current around these
potentials (16), thus contributing to setting the resting membrane
potential and to subthreshold electrogenesis (18). Heterologous
expression of recombinant rNav1.9a in HEK293 mammalian
cells or DRG neurons in culture for 7 days after biolistic
transformation did not show TTX-R currents similar to those observed
in vivo.2 One
possibility is that these expression systems may lack a factor(s) that
is important for the stability or efficient anchoring of the
rNav1.9a channels in the membranes of these cells or the
modulation of these channels so that they can produce the predicted
Na+ currents. The expression of the Drosophila
sodium channel para in heterologous expression
systems is dependent upon the co-expression of tipE factor
(49).
-subunit of Na+/K+-ATPase in this
pathway (55, 56). It is not unreasonable to speculate that
Nav1.9a may have a function other than the generation of a
persistent sodium current in DRG neurons (16) and that it may
participate in the export of FHF1B to the cell surface or the exterior
of the cell (55, 56), where it might exert its function. Alternatively,
the interaction between rFHF1B and the C terminus of
rNav1.9a may modulate this channel directly or indirectly
by recruiting other proteins to the channel complex.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Joel A. Black and William N. Hormuzdiar for providing DRG cultures, Dr. Ted Cummins for helpful discussions, and Bart Toftness for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by grants from the National Multiple Sclerosis Society and the Rehabilitation Research and Development Service and Medical Research Services, Department of Veterans Affairs and by gifts from the Paralyzed Veterans of America and Eastern Paralyzed Veterans Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF348446.
Supported by a Spinal Cord Research Fellowship from the
Eastern Paralyzed Veterans Association.
** To whom correspondence should be addressed: PVA/EPVA Neuroscience Research Center, Yale University School of Medicine, 127A, Bldg. 34, 950 Campbell Ave., West Haven, CT 06516. Tel.: 203-937-3802; Fax: 203-937-3801; E-mail: sulayman.dib-hajj@yale.edu.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M101606200
2 S. D. Dib-Hajj, T. R. Cummins, L. Tyrrell, and S. G. Waxman, unpublished information.
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ABBREVIATIONS |
---|
The abbreviations used are: DRG, dorsal root ganglia; TTX, tetrodotoxin; TTX-R, tetrodotoxin-resistant; Y2H, yeast two-hybrid screening; FGF, fibroblast growth factor; FHF, FGF homologous factor; GFP, green fluorescent protein; HEK cells, human embryonic kidney cells; aa, amino acids; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; DBD, DNA binding domain; AP, activation domain; UR, unique region; CR, conserved region; SD, synthetic dropout base.
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REFERENCES |
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---|
1. | Goldin, A. L., Barchi, R. L., Caldwell, J. H., Hofmann, F., Howe, J. R., Hunter, J. C., Kallen, R. G., Mandel, G., Meisler, M. H., Netter, Y. B., Noda, M., Tamkun, M. M., Waxman, S. G., Wood, J. N., and Catterall, W. A. (2000) Neuron 28, 365-368[Medline] [Order article via Infotrieve] |
2. | Beckh, S., Noda, M., Lübbert, H., and Numa, S. (1989) EMBO J. 8, 3611-3616[Abstract] |
3. |
Dumaine, R.,
Wang, Q.,
Keating, M. T.,
Hartmann, H. A.,
Schwartz, P. J.,
Brown, A. M.,
and Kirsch, G. E.
(1996)
Circ. Res.
78,
916-924 |
4. | Cannon, S. C. (1997) Neuromuscul. Disord. 7, 241-249[CrossRef][Medline] [Order article via Infotrieve] |
5. | Barchi, R. L. (1995) Annu. Rev. Physiol. 57, 355-385[CrossRef][Medline] [Order article via Infotrieve] |
6. | Ptacek, L. J. (1997) Neuromuscul. Disord. 7, 250-255[CrossRef][Medline] [Order article via Infotrieve] |
7. | Rizzo, M. A., Kocsis, J. D., and Waxman, S. G. (1996) Eur. Neurol. 36, 3-12[CrossRef][Medline] [Order article via Infotrieve] |
8. | Waxman, S. G., Dib-Hajj, S., Cummins, T. R., and Black, J. A. (2000) Brain Res. 886, 5-14[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Waxman, S. G.,
Dib-Hajj, S.,
Cummins, T. R.,
J.,
and Black, J. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7635-7639 |
10. |
Gee, S. H.,
Madhavan, R.,
Levinson, S. R.,
Caldwell, J. H.,
Sealock, R.,
and Froehner, S. C.
(1998)
J. Neurosci.
18,
128-137 |
11. |
Cantrell, A. R.,
Scheuer, T.,
and Catterall, W. A.
(1999)
J. Neurosci.
19,
5301-5310 |
12. |
Tibbs, V. C.,
Gray, P. C.,
Catterall, W. K.,
and Murphy, B. J.
(1998)
J. Biol. Chem.
273,
25783-25788 |
13. | Ratcliffe, C. F., Qu, Y., McCormick, K. A., Tibbs, V. C., Dixon, J. E., Scheuer, T., and Catterall, W. A. (2000) Nat. Neurosci. 3, 437-444[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Dib-Hajj, S. D.,
Tyrrell, L.,
Black, J. A.,
and Waxman, S. G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8963-8968 |
15. | Tate, S., Benn, S., Hick, C., Trezise, D., John, V., Mannion, R. J., Costigan, M., Plumpton, C., Grose, D., Gladwell, Z., Kendall, G., Dale, K., Bountra, C., and Woolf, C. J. (1998) Nat. Neurosci. 1, 653-655[CrossRef][Medline] [Order article via Infotrieve] |
16. | Cummins, T. R., Dib-Hajj, S. D., Black, J. A., Akopian, A. N., Wood, J. N., and Waxman, S. G. (1999) J. Neurosci. 19, RC43[Medline] [Order article via Infotrieve] |
17. | Dib-Hajj, S. D., Tyrrell, L., Cummins, T. R., Black, J. A., Wood, P. M., and Waxman, S. G. (1999) FEBS Lett. 462, 117-120[CrossRef][Medline] [Order article via Infotrieve] |
18. | Herzog, R. I., Cummins, T. R., and Waxman, S. G. (2000) Soc. Neurosci. Abstr. 26, 1111 |
19. | Fields, S., and Song, O. (1989) Nature 340, 245-246[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Nehring, R. B.,
Wischmeyer, E.,
Doring, F.,
Veh, R. W.,
Sheng, M.,
and Karschin, A.
(2000)
J. Neurosci.
20,
156-162 |
21. | Braithwaite, S. P., Meyer, G., and Henley, J. M. (2000) Neuropharmacology 39, 919-930[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Roche, K. W.,
Ly, C. D.,
Petralia, R. S.,
Wang, Y. X.,
McGee, A. W.,
Bredt, D. S.,
and Wenthold, R. J.
(1999)
J. Neurosci.
19,
3926-3934 |
23. | Mori, M., Konno, T., Ozawa, T., Murata, M., Imoto, K., and Nagayama, K. (2000) Biochemistry 39, 1316-1323[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Smallwood, P. M.,
Munoz-Sanjuan, I.,
Tong, P.,
Macke, J. P.,
Hendry, S. H.,
Gilbert, D. J.,
Copeland, N. G.,
Jenkins, N. A.,
and Nathans, J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9850-9857 |
25. | Fjell, J., Hjelmstrom, P., Hormuzdiar, W., Milenkovic, M., Aglieco, F., Tyrrell, L., Dib-Hajj, S., Waxman, S. G., and Black, J. A. (2000) Neuroreport 11, 199-202[Medline] [Order article via Infotrieve] |
26. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1994) Current Protocols in Molecular Biology , pp. 4.2.4-4.2.6, John Wiley & Sons, Inc., New York |
27. | Dib-Hajj, S. D., Hinson, A. W., Black, J. A., and Waxman, S. G. (1996) FEBS Lett. 384, 78-82[CrossRef][Medline] [Order article via Infotrieve] |
28. | Hollenberg, S. M., Sternglanz, R., Cheng, P. F., and Weintraub, H. (1995) Mol. Cell. Biol. 15, 3813-3822[Abstract] |
29. | Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205-214[Medline] [Order article via Infotrieve] |
30. |
Liu, C. J.,
Wang, H.,
and Lengyel, P.
(1999)
EMBO J.
18,
2845-2854 |
31. | Voit, R., Schafer, K., and Grummt, I. (1997) Mol. Cell. Biol. 17, 4230-4237[Abstract] |
32. | Lee, W. S., Kao, C. C., Bryant, G. O., Liu, X., and Berk, A. J. (1991) Cell 67, 365-376[Medline] [Order article via Infotrieve] |
33. |
Liu, C.,
Wang, H.,
Zhao, Z., Yu, S.,
Lu, Y. B.,
Meyer, J.,
Chatterjee, G.,
Deschamps, S.,
Roe, B. A.,
and Lengyel, P.
(2000)
Mol. Cell. Biol.
20,
7024-7036 |
34. |
Wang, H.,
Liu, C.,
Lu, Y.,
Chatterjee, G.,
Ma, X. Y.,
Eisenman, R. N.,
and Lengyel, P.
(2000)
J. Biol. Chem.
275,
27377-27385 |
35. | Jeong, S. Y., Goto, J., Hashida, H., Suzuki, T., Ogata, K., Masuda, N., Hirai, M., Isahara, K., Uchiyama, Y., and Kanazawa, I. (2000) Biochem. Biophys. Res. Commun. 267, 262-270[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Munoz-Sanjuan, I.,
Simandl, B. K.,
Fallon, J. F.,
and Nathans, J.
(1999)
Development
126,
409-421 |
37. | Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262[CrossRef][Medline] [Order article via Infotrieve] |
38. | Felts, P. A., Yokoyama, S., Dib-Hajj, S., Black, J. A., and Waxman, S. G. (1997) Mol. Brain Res. 45, 71-82[CrossRef][Medline] [Order article via Infotrieve] |
39. | Isom, L. L., Ragsdale, D. S., De Jongh, K. S., Westenbroek, R. E., Reber, B. F., Scheuer, T., and Catterall, W. A. (1995) Cell 83, 433-442[Medline] [Order article via Infotrieve] |
40. | Isom, L. L., De Jongh, K. S., Patton, D. E., Reber, B. F., Offord, J., Charbonneau, H., Walsh, K., Goldin, A. L., and Catterall, W. A. (1992) Science 256, 839-842[Medline] [Order article via Infotrieve] |
41. |
Patton, D. E.,
Isom, L. L.,
Catterall, W. A.,
and Goldin, A. L.
(1994)
J. Biol. Chem.
269,
17649-17655 |
42. |
Srinivasan, J.,
Schachner, M.,
and Catterall, W. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15753-15757 |
43. |
Xiao, Z. C.,
Ragsdale, D. S.,
Malhotra, J. D.,
Mattei, L. N.,
Braun, P. E.,
Schachner, M.,
and Isom, L. L.
(1999)
J. Biol. Chem.
274,
26511-26517 |
44. |
Qu, Y.,
Rogers, J. C.,
Chen, S. F.,
McCormick, K. A.,
Scheuer, T.,
and Catterall, W. A.
(1999)
J. Biol. Chem.
274,
32647-32654 |
45. |
McCormick, K. A.,
Srinivasan, J.,
White, K.,
Scheuer, T.,
and Catterall, W. A.
(1999)
J. Biol. Chem.
274,
32638-32646 |
46. |
Sampo, B.,
Tricaud, N.,
Leveque, C.,
Seagar, M.,
Couraud, F.,
and Dargent, B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3666-3671 |
47. |
Cummins, T. R.,
Black, J. A.,
Dib-Hajj, S. D.,
and Waxman, S. G.
(2000)
J. Neurosci.
20,
8754-8761 |
48. | Fjell, J., Cummins, T. R., Dib-Hajj, S. D., Fried, K., Black, J. A., and Waxman, S. G. (1999) Mol. Brain Res. 67, 267-282[CrossRef][Medline] [Order article via Infotrieve] |
49. | Feng, G., Deak, P., Kasbekar, D. P., Gil, D. W., and Hall, L. M. (1995) Cell 82, 1001-1011[Medline] [Order article via Infotrieve] |
50. |
Munoz-Sanjuan, I.,
Smallwood, P. M.,
and Nathans, J.
(2000)
J. Biol. Chem.
275,
2589-2597 |
51. | Verdier, A. S., Mattei, M. G., Lovec, H., Hartung, H., Goldfarb, M., Birnbaum, D., and Coulier, F. (1997) Genomics 40, 151-154[CrossRef][Medline] [Order article via Infotrieve] |
52. | Coulier, F., Pontarotti, P., Roubin, R., Hartung, H., Goldfarb, M., and Birnbaum, D. (1997) J. Mol. Evol. 44, 43-56[Medline] [Order article via Infotrieve] |
53. | Hartung, H., Feldman, B., Lovec, H., Coulier, F., Birnbaum, D., and Goldfarb, M. (1997) Mech. Dev. 64, 31-39[CrossRef][Medline] [Order article via Infotrieve] |
54. | Florkiewicz, R. Z., Majack, R. A., Buechler, R. D., and Florkiewicz, E. (1995) J. Cell. Physiol. 162, 388-399[Medline] [Order article via Infotrieve] |
55. |
Florkiewicz, R. Z.,
Anchin, J.,
and Baird, A.
(1998)
J. Biol. Chem.
273,
544-551 |
56. | Trudel, C., Faure-Desire, V., Florkiewicz, R. Z., and Baird, A. (2000) J. Cell. Physiol. 185, 260-268[CrossRef][Medline] [Order article via Infotrieve] |