From the Department of Medicine, The Johns Hopkins
University School of Medicine and the
Department of Molecular
Biology and Genetics, The Johns Hopkins University,
Baltimore, Maryland 21205, the ** Dana-Farber Cancer
Institute, the
Department of Medicine,
Brigham and Women's Hospital, the Department of Pediatrics,
Children's Hospital of Boston, and Harvard Medical School,
Boston, Massachusetts 02115, and the ¶ Department of Medicine,
University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Received for publication, November 22, 2000, and in revised form, March 22, 2001
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ABSTRACT |
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A complex family of 4.1R isoforms has been
identified in non-erythroid tissues. In this study we characterized the
exonic composition of brain 4.1R-10-kDa or
spectrin/actin binding (SAB) domain
and identified the minimal sequences required to stimulate fodrin/F-actin association. Adult rat brain expresses predominantly 4.1R mRNAs that carry an extended SAB, consisting of the
alternative exons 14/15/16 and part of the constitutive exon 17. Exon
16 along with sequences carried by exon 17 is necessary and sufficient to induce formation of fodrin-actin-4.1R ternary complexes. The ability
of the respective SAB domains of 4.1 homologs to sediment fodrin/actin
was also investigated. 4.1G-SAB stimulates association of fodrin/actin,
although with an ~2-fold reduced efficiency compared with
4.1R-10-kDa, whereas 4.1N and 4.1B do not. Sequencing of the
corresponding domains revealed that 4.1G-SAB carries a cassette that
shares significant homology with 4.1R exon 16, whereas the respective
sequence is divergent in 4.1N and absent from brain 4.1B. An ~150-kDa
4.1R and an ~160-kDa 4.1G isoforms are present in PC12 lysates that
occur in vivo in a supramolecular complex with fodrin and
F-actin. Moreover, proteins 4.1R and 4.1G are distributed underneath
the plasma membrane in PC12 cells. Collectively, these observations
suggest that brain 4.1R and 4.1G may modulate the membrane mechanical
properties of neuronal cells by promoting fodrin/actin association.
Erythroid protein 4.1R is an 80-kDa phosphoprotein that plays a
pivotal role in the structural organization and maintenance of the red
blood cell cytoskeleton (1). Specifically, it stabilizes the
spectrin-actin complexes (2-5) and mediates the attachment of the
underlying cytoskeleton to the overlying lipid bilayer through
interactions with integral membrane proteins, such as band 3 (6) and
glycophorin C (7, 8). Abnormalities in 4.1R are associated with
congenital red cell defects leading to severe membrane fragmentation
and hereditary elliptocytosis (9).
The major functions of erythroid 4.1R have been well described. In
brief, the 30-kDa NH2-terminal domain interacts with
transmembrane proteins including glycophorin C, calmodulin, p55, and
band 3 (6-8), whereas the 10-kDa or
SAB1 domain is required for
the formation and stabilization of spectrin-actin complexes (10, 11).
In the erythrocyte system, the SAB domain is encoded by the
alternatively spliced exon 16 (63 nts/21 amino acids) and the majority
of the constitutive exon 17 (135 nts/45 amino acids out of 177 nts)
(12, 13). The spectrin/actin/4.1R interactions have been extensively
studied in red blood cells, indicating that inclusion of exon 16 along
with amino acid residues carried by exon 17 are critical for
spectrin/4.1R binary association, actin/4.1R direct interaction, and
spectrin-actin-4.1R ternary complex formation (11).
During the past 10 years, it has been shown that the prototypical
erythroid 4.1R is only one of the multiple isoforms that arise from the
single 4.1R gene through extensive alternative pre-mRNA
splicing (14-17). Consequently, discrete internal sequence motifs are
preferentially included or omitted in different non-erythroid tissues
and organs, giving rise to a complex family of 4.1R modular isoforms.
Moreover, three novel 4.1-like genes were recently added to the already
complex NF2/ERM/4.1 gene family, namely
4.1G, 4.1N, and 4.1B (18). 4.1G
is widely expressed among different tissues and organs (19), whereas
4.1N and 4.1B appear to be confined in the peripheral and central
neurons and the brain, respectively (19-24). A significant degree of
homology within the 30-, 10-, and 22/24-kDa domains is shared by 4.1R
and the three 4.1-like genes, whereas the NH2 terminus and
intervening sequences appear diverse and highly specific. Specifically,
a 73% identity is shared between 4.1R- and 4.1G-SAB domains (19),
whereas a 36% similarity exists between the respective 4.1R and 4.1N
domains (22). Furthermore, brain 4.1B exhibits an ~50% homology
within the COOH-terminal 45 residues of 4.1R-10-kDa carried by exon 17 (23, 24).
Spectrin is a major component of the erythrocyte cytoskeleton, where it
plays an essential role in the maintenance of the mechanical stability
and shape of the cell membrane (25). Erythroid spectrin consists of two
subunits, encoded by Initial observations by Burns et al. (35) indicated that
fodrin, F-actin, and erythroid-4.1R form a ternary complex, linking the
fodrin tetramers into a hexagonal lattice. Furthermore, Tyler et
al. (36) and Liu et al. (37) demonstrated that
In the present study we endeavored to decipher the minimal sequences
within brain 4.1R-SAB domain required for fodrin-actin-4.1R ternary
complex formation. Moreover, we assessed the ability of the respective
domains of the 4.1-homologs, 4.1G, 4.1N, and 4.1B, to induce
fodrin/actin association in vitro. Finally, we investigated the potential in vivo association of 4.1 proteins with
fodrin tetramers and actin in the neuronal PC12 cell line. Our results suggest that brain 4.1R and 4.1G may be essential for the maintenance of the shape and membrane structural integrity of neural cells, through
their involvement in the formation and/or stabilization of the
fodrin-actin complexes. To the contrary, 4.1N and 4.1B appear to have
distinct functional activities in neuronal tissues from stabilizing the
cytoskeletal fodrin/actin network.
RNA Isolation and RT-PCR Analysis of Non-erythroid 4.1R-SAB
Domain--
Mouse and rat tissue samples were dissected after
perfusion of the animals via the left ventricular ascending aorta with
1× PBS, immediately frozen in liquid N2, and ground to
fine powder. Total RNA from several tissues and two cell lines (PC12
and MOLT4) was prepared using Trizol Reagent according to the
manufacturer's instructions (Life Technologies, Inc.). Subsequently, 5 µg of total RNA were subjected to reverse transcription-polymerase
chain reaction (RT-PCR) following standard experimental protocols. The primers used in the PCR amplification reaction were chosen to flank the
4.1R-SAB domain and were as follows: primer a (within exon 13), nts
1815-1847,
5'-AAAGGTACCATCGATGCTGTCGATTCGGCAGACCGAAGTCCTCGGCCC-3' (sense) and primer b (located in exon 17), nts 2321-2351,
5'-AAAGGTACCATCGATTCCTGTGGGGATTTGCCCATTGATGTTAAG-3' (antisense) (17).
Subsequently, the PCR products were subcloned into pGEM-4Z (Promega,
San Luis Obispo, CA), and a number of clones obtained from separate PCR
amplifications were analyzed by sequencing (T7 Sequenase version 2.0 kit, U. S. Biochemical Corp., Cleveland, OH).
Generation of 4.1R, 4.1G, 4.1N, and 4.1B GST Fusion Proteins and
Bacterial Expression--
GST-4.1R-10-kDa peptides, shown in Fig. 2,
A and B, were prepared by PCR amplification using
a rat brain cDNA clone as template and the following sets of
primers: (i) primers a and b; (ii) primer c,
5'-ATGGATCCAAGGATTTAGACAAGAGTCA-3', and primer g,
5'-ATGAATTCGAAGGGTGAGTGTGTGGACA-3'; (iii) primer d,
TAGGATCCAAGGTGGAAAAACCCCACAC-3', and primer g; (iv) primer e,
5'-TAGGATCCAAGAAGCTTGCAGAAAAAGG-3', and primer g; and (v) primer f,
5'-TAGGATCCAAGAAAAAGAGAGAGAGACT-3', and primer g. Similarly, the
4.1R-SAB domain deletion peptides, shown in Fig. 3A, were
generated with the use of the sense primer d in combination with the
antisense primers g and h, 5'-GTCCCACTCGCTGGGCCGTGGTT-3'; primer i,
5'-TGATTCCATAAAGTTCTTTTTCAG-3'; or primer j,
5'-GATGCTGGCATGATGCTTCTTGA-3'. A two-step PCR approach was used to
generate the lysine deletion construct with the set of primers d and k,
5'-AGTTCTTCAGTTCACTGATGCTGGCATGATG-3' (antisense), and primer l,
5'-AACTGAAGAACTTTATGGAATCAGT-3', (sense) and primer g. To produce the
respective 4.1G, 4.1N, and 4.1B SAB domains, RT-PCR of total RNA,
obtained from rat brain, was employed using the following sets of
primers: for amplification of 4.1G-10-kDa, 5'-ACGAATTCGGAAAGAAAAATACCTTGAGA-3' (sense) and
5'-ACGTCGACGAGAGGCAGAGGTGTGACCCG-3' (antisense); for production of
4.1N-10-kDa, 5'-ACGAATTCACTAAGATCAAGGAGCTGAAG-3' (sense) and
5'-ACGTCGACTGAGGGCAGCCTGCGCTCCCG-3' (antisense); and for generation of
4.1B-10-kDa, 5'-ACGAATTCGCAGCTGATGGAGAGACCAGT-3' (sense) and
5'-ACGTCGACCACGGGAGATGTAGAGAGCCT-3' (antisense). Subsequently, the amplified fragments were subcloned into the bacterial expression vector pGEX-2T (Amersham Pharmacia Biotech) and verified by sequencing (T7 Sequenase version 2.0 kit, U. S. Biochemical Corp.). The
recombinant polypeptides were overexpressed by
isopropyl-1-thio-
To obtain the complete sequence of brain 4.1G-, 4.1N-, and 4.1B-10-kDa
domains (Fig. 4A) the following primer sets that were flanking the respective SAB domains were used: 4.1G-sense
5'-CCCCTGCCCGCTGAGGGA-3', 4.1N-sense 5'-GAGGTCAGGACGCCCACT-3', and
4.1B-sense 5'-CACACCTGTCACAGCCCT-3', in combination with the antisense
primer 5'-TCCCTTCACAGTTTTGGT-3'. The accession numbers of 4.1G, 4.1N,
and 4.1B cDNAs are AF044312, AB019256, and AAD38048, respectively.
Antibody Production--
Synthetic peptides
N-EKTHIEVTVPTSNGD-C, N-KLAEKTEDLIRMR-C, and N-KKKRERLDGENIYIR-C,
corresponding to 4.1R-exons 14, 15, and 16, respectively, were
used as immunogens for generation of polyclonal antibodies (Ab). The
anti-Exon14, anti-Exon15, and anti-Exon16 antibodies were subsequently
affinity-purified using the Sulfolink-kit according to manufacturer's
instructions (Pierce). The 4.1R anti-16-kDa antibody was produced and
purified as described previously (14). The polyclonal anti-fodrin Ab
was a generous gift from Dr. R. J. Bloch (University of Maryland)
and was generated against purified bovine brain Purification and Iodination of Rat Brain Fodrin--
Fodrin
purification was carried out as described previously (41). In brief,
rat brains (~10 g) were dissolved in homogenization buffer (10 mM imidazole HCl, 5 mM EDTA, 3 mM
NaN3, 1 mM EGTA, pH 7.3) after removal of the
meninges and major blood vessels. Fodrin was extracted in a high salt
buffer (10 mM Tris-HCl, 50 mM KCl, 3 mM NaN3, 1 mM MgCl2,
0.1 mM CaCl2, 0.1 mM DTT, pH 8.2), (NH4)2SO4-precipitated, and
gel-filtrated through Sepharose 4B-CL, previously equilibrated with
TKE/KCl (10 mM Tris-HCl, 700 mM KCl, 0.5 mM EDTA, 0.1 mM DTT, pH 8.2). Subsequently, the
fodrin-rich fractions, as assessed by 8% SDS-PAGE, were combined and
followed by a second (NH4)2SO4
precipitation, dialysis against a low salt precipitation buffer (10 mM Tris-HCl, 3 mM NaN3, 0.1 mM CaCl2, 0.1 mM DTT, pH 8.2), and
sedimentation through a linear sucrose gradient (5-20%) in TKE/KCl
buffer. The pooled fractions from the sucrose gradient were dialyzed
either against storage buffer (1 mM
Na2HPO4, 3 mM NaN3, 0.1 mM DTT, pH 8.0) or binding buffer (130 mM KCl,
25 mM NaCl, 2 mM MgCl2, 0.4 mM DTT, 0.2 mM CaCl2, 20 mM Hepes, pH 7.2) (42). Ultrafiltration with Centriprep-100 (Amicon Inc., Beverly, MA) was used to concentrate the fodrin preparation, and the purity of the protein was assayed by 8% SDS-PAGE. In all purification steps 1 mM phenylmethylsulfonyl
fluoride protease inhibitor was included.
Iodination of brain fodrin was essentially carried out as described
(43). Briefly, 30 µl of purified fodrin (4 µg/µl) was incubated
in an equal volume of reaction buffer (100 mM Tris-HCl, 0.025% NaN3, pH 7.5) plus 10 µl of 0.2% chloramine T in
the presence of 5 µl (0.5mCi) of 125I (Amersham Pharmacia
Biotech) for 2 min on ice. To stop the reaction, 40 µl of in
situ prepared 1% Na2S2O5 and
40 µl of 1% KI were added sequentially. Iodinated fodrin was
extensively dialyzed at 4 °C against binding buffer to remove any
unreacted label with the use of Dispo-Biodialyzers (Amika Corp.,
Columbia, MD).
Co-sedimentation Assays and Quantitative
Studies--
Commercially obtained muscle G-actin was polymerized at a
concentration of 1 mg/ml, according to the manufacturer's instructions (Cytoskeleton, Denver, CO). Purified brain fodrin, recombinant GST
fusion polypeptides, and F-actin were subsequently dialyzed against
several changes of binding buffer at 4 °C. Co-sedimentation assays
were performed in 50 µl of binding buffer in the presence of 0.2 µM fodrin, 5.7 µM F-actin, and 1.4 µM either of GST-4.1R, GST-4.1G, GST-4.1N, GST-4.1B, or
GST-alone polypeptides. The reaction mixtures were incubated at room
temperature for 45 min and then centrifuged at 4 °C for 1 h at
100,000 × g in a Beckman 42.2Ti rotor. Equivalent
portions of supernatants and pellets were subsequently fractionated by
10% SDS-PAGE.
For quantitative binding studies 0.2 µM radioiodinated
fodrin was allowed to interact with 5.7 µM F-actin in the
presence of increasing amounts of either recombinant GST-4.1R-10-kDa or GST-4.1G-10-kDa polypeptides (0.14-1.4 µM). The pelleted
protein complexes were subsequently counted in a Cell Culture and Confocal Microscopy--
Rat PheoChromocytoma
cells (PC12) (21) were grown on poly-D-lysine-coated
coverslips in RPMI supplemented with 5% fetal bovine serum, 10% horse
serum, and 100 units of penicillin/streptomycin, at 37 °C with 5%
CO2 atmosphere in a humidified incubator. Adhered cells
were washed 3× with PBS and fixed in freshly prepared 4% paraformaldehyde in PBS for 20 min at room temperature. Subsequently, cells were rinsed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature. Unreactive groups were
blocked by incubation with 10% normal goat serum in PBS for 1 h
at room temperature. In single labeling experiments the following primary antibodies were utilized: anti-Exon16 (4.1R, 1:100), anti-4.1G (1:200), and anti-fodrin in PBS plus 3 mg/ml bovine serum albumin incubated at 37 °C for 2 h. Following extensive washes in PBS, cells were counterstained either with goat anti-rabbit fluorescein isothiocyanate or goat anti-rabbit Texas Red IgGs (1:50, Pierce) at
37 °C for 1 h and extensively washed with PBS. In double
labeling experiments, after the first set of staining, PC12 cells were blocked for 8 h at 4 °C with 10 µg/ml unconjugated goat
anti-rabbit Fab fragment (Jackson ImmunoResearch Laboratories, Inc.,
West Grove, PA) in PBS and then processed for the second set of
immunolabeling. Subsequently, coverslips were mounted with
Vectashield (Vector Laboratories, Burlingame, CA) and analyzed
either under a Nikon microphot FXA microscope (Garden City, NY) or a
laser scanning confocal system (Noran Instruments, Middleton, WI)
combined with a Zeiss Axiophot microscope (Thornwood, NY) through a
100× oil immersion objective.
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
assays were performed using PC12 cell extracts as described previously
(44) with some modifications. About 4 × 106 cells
were collected by scraping with a rubber policeman, washed 3× in
ice-cold PBS, resuspended in lysis buffer (50 mM Tris-HCl, 125 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium
deoxycholate, 0.2 mM EDTA, 5 mM
iodoacetamide, pH 7.5) plus protease inhibitors (39), and given
20 strokes in a tight-fitting glass homogenizer. The lysate was
centrifuged at 14,000 × g for 20 min at
4 °C, and the supernatant was collected. Protein content was
determined using a standard Bradford assay (Pierce), and 750 µg of
total lysate were precleared with 150 µl of a 50% suspension of
protein A-Sepharose-6MB beads (Amersham Pharmacia Biotech) on a 4 °C
rocker for 2 h. Pre-immune serum, anti-Exon16, and anti-fodrin
antibodies, containing 5 µg of IgGs, were allowed to interact with
150 µl of protein A-Sepharose-6MB in the presence of 1× PBS at
4 °C with gentle rocking for 4 h. Subsequently, the antibodies
bound to the beads were incubated with the precleared PC12 extracts in a rocking platform at 4 °C for 6-8 h. Following the end of the incubation period, the samples were centrifuged for 15-20 s at 14,000 × g at 4 °C. The supernatants were collected
and stored at Alternative Splicing Events within the 4.1R-SAB Domain of
Non-erythroid Tissues--
To characterize the splicing patterns
within the non-erythroid 4.1R-SAB domain, total RNA from several
tissues of rat or mouse origin was subjected to RT-PCR analysis
followed by sequencing. Three alternatively spliced cassettes encoded
by exons 14 (57 nts), 15 (42 nts), and 16 (63 nts) along with the
majority of the constitutive exon 17 (135 out of 177 nts) compose the
non-erythroid 4.1R-10-kDa. These exhibited complex splicing patterns
resulting in the generation of multiple combinatorial products that
were expressed in variable amounts among different non-erythroid
tissues and cell lines (Fig.
1A). Interestingly, adult rat
brain and undifferentiated PC12 cells contained a substantial amount of
a bigger size PCR product that included all three alternatively spliced
exons 14-16, although additional PCR bands were also detected that
corresponded to different exonic combinations. Sequence comparison of
exons 14-16 from several species, including human, mouse, rat, and
bovine, revealed extensive evolutionary conservation of these motifs
with only few nucleotide changes (Fig. 1B).
Sequences within Exons 16 and 17 Are Necessary and Sufficient to
Promote Fodrin-Actin-4.1R Ternary Complex Formation--
To identify
the exons within the 4.1R-SAB required for stimulation of
fodrin-actin-4.1R ternary complex formation, we generated various
GST-4.1R-10-kDa recombinant peptides that carried distinct combinations
of the alternative spliced cassettes 14-16 (Fig. 2A). The authenticity of the
resultant fusion polypeptides was verified by immunoblot assays with
the use of appropriate anti-4.1R antibodies (Fig. 2B).
Subsequently, the ability of recombinant GST-4.1R-10-kDa variants to
promote fodrin/actin association was tested in a series of
co-sedimentation assays (Fig. 2C). In all cases in which
4.1R-10-kDa peptides carried the alternatively spliced exon 16 along
with the constitutive 45 residues carried by exon 17, a significant, almost complete, shift of fodrin from the supernatant to the pellet fraction was observed either in the presence or absence of exons 14 and
15. On the contrary, when the 63-nt cassette encoded by exon 16 was
omitted, almost equivalent amounts of fodrin were detected in both
supernatant and pellet fractions. These results were similar to control
experiments in which the 4.1R-10-kDa was omitted. Thus, the 4.1R-SAB
domain stimulates co-sedimentation of fodrin and F-actin in an exon
16-dependent manner, regardless of the presence of the
nucleotide motifs encoded by the alternatively spliced cassettes 14 and
15. It is of interest to note that the combination of exons 14 and 15 exhibits stronger activity in stimulating the sedimentation of fodrin
and actin even though the individual exon appears to suppress the
binding when present alone. A graphic presentation of the percentage of
pelleted fodrin following stimulation with the various 4.1R-10-kDa
peptides is shown in Fig. 2D after quantitation of the
respective bands using NIH Image Software.
To determine the minimal sequences within exon 17 necessary to promote
fodrin-actin-4.1R ternary complex formation, we generated GST fusion
peptides containing all three alternative exons 14/15/16 and varying
lengths of the constitutive 45 residues that belong to exon 17. Specifically, three constructs were produced that either included the
first 36, 26, or 16 NH2-terminal amino acids of exon 17 (Fig. 3A). Subsequent
co-sedimentation assays demonstrated that recombinant polypeptides
carrying any COOH-terminal deletion either completely failed
(i.e. 10kd-26 and 10kd-16 constructs) or showed very limited
ability (i.e. 10kd-36 peptide) to induce fodrin/actin
association (Fig. 3B). These observations indicated that the
last 10 COOH-terminal residues of 4.1R-SAB, within exon 17, are also
critical in the formation of fodrin-actin-4.1R ternary complexes.
Moreover, similar results were obtained when 4.1R GST fusion peptides
carrying exon 16, but skipping exons 14 and 15, along with various
COOH-terminal deletions of exon 17 (e.g. 10kd/Ex16, 10kd/Ex16-36 and 10kd/Ex16-26) were examined in a co-sedimentation assay (Fig. 3C).
Furthermore, a case of heterozygous hereditary elliptocytosis
associated with partial deficiency of 4.1R has been reported in nine
related French families (45). Molecular genetic analysis revealed a
single codon deletion in the 4.1R locus resulting in elimination of a
lysine residue located either at position 447 or 448 within the
4.1R-10-kDa domain. The abnormal 4.1R protein that is produced fails to
interact with spectrin-actin complexes within the erythrocyte.
Consequently, we generated by a two-step PCR approach GST-4.1R-SAB
polypeptides that were missing either of these lysine residues (Fig.
3A, 10kd-Lys), and we investigated their ability to induce
fodrin/actin co-sedimentation. As shown in Fig. 3, B and
C, the sole deletion of lysine 447 (10kd-Lys and
10kd/Ex16-Lys) abolishes ternary complex formation, indicating that
amino acids proven to be important in stimulating formation of
erythroid-4.1R-spectrin-actin complexes are also critical in promoting
brain-4.1R/fodrin/actin association as well. Identical results were
obtained when lysine 448 was omitted (data not shown).
Taken altogether, the results obtained in Figs. 2 and 3 indicate that
sequences carried by both exons 16 and 17 are necessary and sufficient
to stimulate fodrin/actin association.
Brain 4.1G- but Neither 4.1N- Nor 4.1B-SAB Domains May Induce
Fodrin-Actin Complex Formation--
To investigate the functional
ability of the 4.1-homologs to polymerize fodrin and actin into
pelletable polymers, GST fusion polypeptides of brain 4.1G-, 4.1N-, and
4.1B-SAB domains were generated (Fig.
4A) and tested in a
co-sedimentation assay (Fig. 4B). Notably, the 4.1G-SAB
paralog was able to stimulate sedimentation of fodrin-actin complexes,
whereas 4.1N and 4.1B did not. Sequence comparison between 4.1R- and
4.1G-, 4.1N-, and 4.1B minimal SAB domains demonstrated that
4.1G-10-kDa carries a nucleotide cassette that shares significant
homology with 4.1R-exon 16 (76%), whereas the respective sequence is
highly divergent in 4.1N-10-kDa and absent from brain 4.1B-10-kDa (Fig.
5A). Furthermore, the lysine residue located at position 447, shown to be essential in the ability
of erythroid and brain 4.1R-10-kDa to induce fodrin/actin association
(Fig. 4B), is not conserved in the 4.1 homologs but is
replaced either by leucine in 4.1G and 4.1N or methionine in 4.1B (Fig.
5A, arrow).
To compare the efficiency of recombinant 4.1R- and 4.1G-10-kDa domains
to promote fodrin/actin association in vitro, we performed a
series of quantitative co-sedimentation assays using increasing concentrations of either 4.1R- or 4.1G-SAB polypeptides (0.14-1.4 µM) and constant amounts of radioiodinated fodrin (1.4 µM) and filamentous actin (5.7 µM). As
shown in Fig. 5B both 4.1R- and 4.1G-10-kDa peptides induced
fodrin/actin sedimentation in a concentration-dependent manner. However, the prototypical 4.1R-SAB domain exhibited a saturating concentration of 0.28 µM, whereas 4.1G-10-kDa
showed a respective value of 0.56 µM. This finding
indicates that 4.1R may stimulate fodrin/actin association with a
2-fold higher efficiency compared with 4.1G at least in
vitro.
Proteins 4.1R and 4.1G Are Present at Points of Cell-Cell Contact
and Occur in Vivo in a Supramolecular Complex with Fodrin Tetramers in
PC12 Cells--
All the aforementioned studies examined the ability of
4.1R- and 4.1G-SAB domains to promote fodrin-actin complex formation in vitro. We also attempted a series of in vivo
experiments in order to evaluate the significance of the results we
obtained within the context of the cell. Consequently, we analyzed the subcellular distribution of 4.1R and 4.1G by confocal microscopy (Figs.
6 and 7,
respectively) as well as their potential in vivo association
with fodrin polymers by co-immunoprecipitation assays (Fig.
8) in the PC12 neuronal cell line.
As shown in Fig. 6, two different anti-4.1R antibodies raised against
either the alternatively spliced exon 16 (Fig. 6, A and
E) or the 4.1R-16-kDa domain (Fig. 6B) revealed
the presence of 4.1R at the periphery of undifferentiated PC12 cells
and especially concentrated at sites of cell-cell contact. When any of
the primary antibodies was omitted, no specific signal was detected
(Fig. 6C). When an anti-fodrin antibody was utilized, which
can identify both
In order to discern whether native 4.1R can associate in
vivo with fodrin and actin, we did a series of
co-immunoprecipitation assays, utilizing PC12 cell extracts and
anti-4.1R-Ex16, anti-fodrin, or control pre-immune IgGs (Fig. 8). Three
identical blots were examined for the presence of protein 4.1R,
The ability of the 4.1G-SAB domain to induce fodrin-actin complex
formation in vitro (Fig. 4B), even with a 2-fold
reduced efficiency compared with 4.1R-10-kDa (Fig. 5B),
prompted us to investigate the presence of 4.1G in the anti-fodrin
immunoprecipitates (Fig. 8D). Indeed an ~160-kDa
immunoreactive band was detected that corresponds to 4.1G, indicating
that 4.1G may also interact in vivo with fodrin tetramers.
However, when the same immunoprecipitates were tested for the presence
of 4.1N and 4.1B, the respective proteins were absent (Fig. 8,
E and F, respectively). The presence of 4.1G and
the absence of 4.1N and 4.1B proteins from the fodrin immunoprecipitates are consistent with the results obtained from our
in vitro co-sedimentation studies (Fig. 4B).
In the current study we identified the structural requirements of
brain 4.1R-SAB domain for ternary complex formation with Characterization of the 4.1R-SAB domain among various non-erythroid
tissues demonstrated the presence of multiple combinatorial products.
In the case of adult rat brain and undifferentiated PC12 cells, a major
mRNA isoform that included the alternatively spliced cassettes
14-16 was identified. Elucidation of the binding behavior of a nested
series of truncated 4.1R-10-kDa peptides revealed that the
alternatively spliced exon 16 along with sequences carried by the
constitutive exon 17 (i.e. the last 10 COOH-terminal amino
acids and lysine 447/448) are necessary and sufficient to promote
fodrin-actin complex formation. These observations suggest that the
minimal sequences of brain 4.1R-SAB required for fodrin/actin association are similar to those of erythroid 4.1R-10-kDa (10, 11) and
argue for functional conservation of the respective domain in diverse
tissues. However, when a potential in vivo association of
4.1R with skeletal muscle spectrin was examined, no such interaction was detected,2 even though
the predominant 4.1R-SAB isotype in striated muscle consists of exons
16 and 17 (44). To the contrary, the skeletal muscle 4.1R-SAB domain
directly interacts in vitro with the major sarcomeric
proteins as follows: myosin heavy chain, Prior studies (46, 47) as well as our findings indicated that fodrin
tetramers are distributed mainly underneath the plasma membrane of
undifferentiated PC12 cells. However, during NGF-induced differentiation fodrin is recruited to perinuclear, spot-like aggregates associated with intermediate filament proteins, including peripherin and neurofilament (46). On the other hand, during PC12
neuronal differentiation, a dramatic down-regulation of 4.1R-SAB mRNA species that include the alternatively spliced exons 14-16 was detected followed by a concomitant increase of a cassette that
contains only the first 45 amino acid residues of the constitutive exon
17 (Fig. 1A, lane PC12+NGF). A possible scenario to explain this differential exonic composition of 4.1R-10-kDa between
undifferentiated and differentiated PC12 cells is that in the former
exon 16 in conjunction with sequences carried by exon 17 may serve to
facilitate and strengthen the formation of fodrin-actin complexes and
thereby rigidify the underlying cortical cytoskeleton. Down-regulation of the expression levels of the minimal fodrin/actin 4.1R-binding domain may permit the redistribution of fodrin and the dynamic reorganization of the cytoskeletal proteins involved in the
morphological differentiation of neurons. This hypothesis is in
agreement with unpublished observations from our
laboratory2 illustrating that upon administration of NGF to
PC12 cells, protein 4.1R departs from the cell membrane and massively
translocates to the nucleus of the differentiated cells, whereas
perinuclear accumulations are also observed that occasionally coincide
with perinuclear fodrin aggregates.
Protein 4.1R shows a very diverse distribution pattern among different
cell types and different phase of the cell cycle. We have demonstrated
that 4.1R is present in the nucleus of interphase and the spindle and
spindle poles of mitotic HeLa cells (39). Moreover, 4.1R is present
both in the nuclear and cytoplasmic compartments of non-confluent
Madin-Darby canine kidney cells, and it is concentrated in the tight
junctions of confluent Madin-Darby canine kidney cells (48). Finally,
4.1R is present both in the cell periphery and in the cytoplasm
exhibiting a rather diffuse staining in PC12 cultures of low density.
However, as cells divide and contact each other, 4.1R accumulates
underneath the plasma membrane and colocalizes with fodrin, at the cell
boundaries. Thus, it becomes obvious that protein 4.1R shuttles
extensively between the nucleus, the cytoplasm, and the cell membrane
depending on the cell type and stage of cell cycle. The trigger or the
mechanisms that allow protein 4.1R to shuttle between the different
cellular compartments have not been identified yet and are now under
investigation in our laboratory. It is reasonable to presume that the
diverse distribution patterns that 4.1R assumes reflect a great
diversity in the functional activities of this protein depending on the developmental stage and the tissue and/or organs that are expressed.
The recent identification of three novel members of the 4.1 gene superfamily (18) prompted us to explore the functional ability of
their respective SAB domain paralogs to promote fodrin/actin association. Our findings indicated that only 4.1G could polymerize fodrin/actin into pelletable polymers, whereas neither 4.1N nor 4.1B
were able to do so. These results are corroborated by the fact that
4.1G-SAB domain carries a nucleotide cassette that shares a significant
degree of homology (76%) with 4.1R-exon 16 (19), whereas the
respective sequence has been highly diversified in 4.1N-10-kDa (22) and
omitted from 4.1B-10-kDa (23, 24). It is noteworthy to mention that
structural characterization of the 4.1B-SAB domain in various adult
mouse tissues has revealed the presence of a cassette that shares 50%
homology with 4.1R-exon 16 in skeletal and cardiac muscle (23), which
is largely skipped in the brain ortholog (Ref. 23 and this work).
Furthermore, the lysine residue, located at position 447 within exon
17, shown to play an essential role in the ability of 4.1R-SAB to
stimulate fodrin/actin co-sedimentation, is replaced either by a
leucine in the cases of 4.1G and 4.1N or a methionine in the case of
4.1B. Interestingly, the lysine residue located at position 448 is
retained in all 4.1 homologs, implying that the positively charged
interface generated by the presence of both lysine residues is critical for fodrin-actin-4.1R ternary complex formation.
When the ability of 4.1R- and 4.1G-SAB domains to promote fodrin-actin
complex formation was evaluated, the prototypical 4.1R-10-kDa demonstrated a 2-fold higher efficiency to stimulate fodrin/actin association, at least in vitro. However, both native 4.1R
and 4.1G proteins appear to occur in vivo in a
supramolecular complex with The presence and subcellular distribution of the diverse 4.1 members
have been extensively studied in brain by several laboratories. 4.1N is
essentially expressed in almost all central and peripheral neurons with
the notable exception of the Purkinje cells of the cerebellum and the
thalamic neurons (21, 22, 50). On the other hand, 4.1B is mostly
enriched in Purkinje cells and thalamic neurons (24). Protein 4.1R also
occurs in the brain where it is distributed in specific neuronal
populations, including the granule cells of the cerebellum and the
dentate gyrus (51). Finally, 4.1G is mainly localized to glia (52).
These observations strongly suggest complementary intracellular
localization patterns among the 4.1-protein members, although
coincident distributions are not precluded. Therefore, distinct
4.1-proteins may possess different binding partners in specific types
of neurons, including components of the neuronal membrane cytoskeleton,
integral membrane channels, and transmembrane receptors. In the case of
4.1R and 4.1G, an in vivo association with
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
I and
I genes, that are aligned side to side to form
heterodimers, which in turn assemble by head to head association to
form tetramers (
I
I)2. In 1981, however, Goodman
et al. (26) demonstrated the presence of spectrin-like
proteins in several non-erythroid cells and tissues. Up until this day,
five distinct genes encoding non-erythroid spectrin-like molecules have
been described in mammals, including
II,
II,
III,
IV, and
V (27-30).
II and
II spectrins have an
apparent molecular mass of ~240 and ~235 kDa, respectively, and are
particularly abundant in brain giving rise to (
II
II)2 brain spectrin or fodrin tetramers (31-34).
-fodrin carries binding sites for protein 4.1R and actin at its
NH2 terminus (reviewed in Ref. 38).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside induction and
purified from bacterial lysate supernatant by glutathione-agarose affinity chromatography. A mixture of protease inhibitors (39) was
included in all peptide preparations. The GST fusion polypeptides were
subjected to 12% SDS-PAGE and either visualized by Coomassie Blue
staining or electrotransferred to polyvinylidene difluoride membrane
for immunoblotting.
-fodrin. It can
recognize both
- and
-fodrin isoforms, whereas reactivity to
erythrocyte
-spectrin was eliminated by pre-adsorption on Sepharose
4B coupled to erythroid
-spectrin (40). Finally, the rabbit
anti-4.1G and anti-4.1N and the goat anti-4.1B IgGs were kindly
provided by Drs. P. Gascard, N. Mohandas, and J. Conboy (University of
California, Berkeley, CA), and a monoclonal anti-
-actin Ab (clone
AC-15) was purchased from Sigma.
-counter, whereas
correction for fodrin/F-actin sedimentation was made by subtracting the
counts pelleted in the absence of GST-4.1R or GST-4.1G peptides. These experiments were conducted in duplicate and repeated twice, producing values with a range difference of
5%.
20 °C, and the beads were extensively washed with
several changes of lysis buffer by rocking at 4 °C. At the end of
the washings the proteins were solubilized in 100 µl of 4×
Laemmli sample buffer and boiled for 5 min. The immunoprecipitates were analyzed by 8% SDS-PAGE and processed for immunoblotting with the
indicated antibodies (see "Results") using an ECL detection kit
(Amersham Pharmacia Biotech).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of the 4.1R-SAB
domain in non-erythroid cells. A, total RNA from
various adult tissues, of mouse (m) or rat (r)
origin, and two different cell lines (MOLT-4 and PC12) was subjected to
RT-PCR amplification with 4.1R-specific primers flanking the 10-kDa
domain (see "Experimental Procedures"). PC12+NGF, PC12
induced to neuronal differentiation in the presence of nerve growth
factor. The exonic composition of major PCR-products is
diagrammatically presented as deduced by sequence analysis of the
obtained bands. B, sequence comparison of the 4.1R-SAB
domain among different species including human, mouse, rat, and bovine
reveals extensive nucleotide conservation; deviant nucleotides are
shown in clear background. M (A/C), Y (C/T), S (C/G), R (A/G), D
(A/G/T), V (A/C/G).
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Fig. 2.
The nucleotide cassette encoded by exon 16 is
required to promote fodrin/actin association. A,
diagrammatic representation of recombinant GST-4.1R-SAB peptides
showing the presence of distinct combinations of the alternatively
spliced exons 14-16. The primer sets used for generation of the
respective products are indicated at the top of each
construct. B, top panel, Coomassie Blue-stained
gel of various GST-4.1R-10-kDa fusion peptides. The authenticity of the
resultant fusion proteins was verified by Western blot analysis using
the appropriate 4.1R antibodies as indicated within individual panels.
C, co-sedimentation assays using the various GST-4.1R-10-kDa
variants (1.4 µM), purified brain fodrin (0.2 µM), and F-actin (5.7 µM). In control
experiments recombinant 4.1R-SAB peptides were either omitted or
replaced by GST protein alone. D, graphic presentation of
the results obtained in (C) using NIH Image Software for
quantitation of the fodrin-band present in the pellet fraction.
S, supernatant; P, pellet.
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Fig. 3.
Sequences within the constitutive exon
17 are necessary to induce fodrin-actin-4.1R ternary complex
formation. A, schematic diagram of
GST-4.1R-10-kDa deletion constructs, carrying either the first 36 (10kd-36), 26 (10kd-26), or 16 (10kd-16) amino acids of the constitutive exon 17. A mutant
4.1R-10-kDa recombinant peptide (Rec. peptides) lacking the
lysine residue located at position 447 within exon 17 is also shown
(10kd-Lys). The primer sets used for amplification of
individual products are depicted. B and C,
co-sedimentation assays using the aforementioned truncated or mutant
4.1R-SAB polypeptides (1.4 µM) containing exons 14/15/16
(B) or exon 16 (C), rat brain fodrin (0.2 µM), and F-actin (5.7 µM). S,
supernatant; P, pellet.
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Fig. 4.
4.1G-10-kDa domain also stimulates
fodrin/actin association in vitro. A,
Coomassie Blue-stained gel of bacterially expressed GST fusion proteins
of rat brain 4.1R-, 4.1G-, 4.1N- and 4.1B-10-kDa domains. B,
co-sedimentation assay using either recombinant 4.1R-, 4.1G-, 4.1N-, or
4.1B-SAB peptides (1.4 µM), brain fodrin (0.2 µM), and F-actin (5.7 µM). S,
supernatant; P, pellet.
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Fig. 5.
The prototypical 4.1R-10-kDa promotes
fodrin/actin association with a 2-fold higher efficiency compared with
4.1G-SAB domain. A, sequence comparison of the minimal
10-kDa domain among the members of the 4.1 gene subfamily.
The arrow points at the lysine residue located at position
447. Domains U2 and U3 correspond to unique
sequences among the members of the 4.1 family (23). In brief, U2
follows the highly conserved membrane binding domain (MBD),
whereas U3 precedes the COOH-terminal 22/24-kDa domain (53). The SAB
domain is situated between U2 and U3 and shares diverse similarity
among the 4.1-homologs, within the alternatively spliced exons 14/15/16
and the constitutive exon 17. The primer sets utilized for production
of the respective 4.1-SAB constructs lie at the boundaries of U2/SAB
(sense primer) and SAB/U3 (antisense primer) and are described in
detail under "Experimental Procedures." B, quantitative
co-sedimentation assays using increasing concentrations of either 4.1R-
or 4.1G-SAB peptides (0.14 -1.4 µM) and constant amounts
of 125I-fodrin (0.2 µM) and filamentous actin
(5.7 µM). Sedimentation of 125I-fodrin
(~0.04 µM) in the absence of 4.1R or 4.1G polypeptides
was subtracted as background. Data points represent average values
obtained from duplicate experiments determined twice with a range
difference of
5%.
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Fig. 6.
Protein 4.1R co-localizes with fodrin at
sites of cell-cell contact in PC12 cells. A-D,
analysis of the intracellular distribution of 4.1R and fodrin in
undifferentiated PC12 cells by immunofluorescent microscopy, using
either 4.1R-specific antibodies: anti-Ex16 (A) and
anti-16-kDa (B) or anti-fodrin (D) IgGs. When the
primary antibodies were omitted no specific signal was detected
(C). The epifluorescent images were obtained under × 40 magnification. E-G, double immunolabeling experiments of
PC12 cells stained with 4.1R anti-Ex16 (E) and anti-fodrin
(F) antibodies, analyzed by confocal microscopy. A
superimposed image of E and F is shown in
G. Confocal images E-G were analyzed under × 100 magnification.
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Fig. 7.
Protein 4.1G is localized underneath the
plasma membrane but is also present within the cytoplasm of PC12
cells. A, confocal microscopy was utilized to analyze
the distribution pattern of 4.1G in cultures of PC12 cells. Protein
4.1G is highly concentrated at points of cell-cell contact
(arrows), whereas significant accumulations were also
observed within the cytoplasm (arrowheads). B, no
specific signal was detected when the primary antibody was eliminated.
Confocal images were obtained under × 100 magnification.
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Fig. 8.
Proteins 4.1R and 4.1G associate in
vivo with -fodrin
tetramers. A-C, co-immunoprecipitation of protein
4.1R, fodrin, and actin from PC12 cell extracts. Anti-4.1R-Ex16
(Ip/Ex16ab) and anti-fodrin antibodies
(IP/fodrin-ab) along with control pre-immune serum
(Ip/PI) were used in the immunoprecipitation assays. The
immunoprecipitates were analyzed by Western blotting either using
anti-Ex16 (A), anti-fodrin (B), or anti-actin
(C) IgGs. D-F, an aliquot of the
anti-fodrin immunoprecipitates was also examined for the presence of
4.1G (D), 4.1N (E), and 4.1B (F) using
its respective antibody. One-fourth of the immunoprecipitates
(Ip) or one-eighth of the supernatant fractions
(Sup) was loaded to the respective lanes.
and
isoforms (40), a rather uniform staining
at the periphery of the cells was detected (Fig. 6, D and
F). Superimposed images of 4.1R (Fig. 6E) and
fodrin (Fig. 6F) revealed complete overlapping at areas of
cell-cell contact (Fig. 6G, areas of co-staining appear
yellow). This finding demonstrated that 4.1R and
-fodrin tetramers co-distribute underneath the plasma membrane in
PC12 cells. Moreover, when PC12 cells were stained for the presence of
4.1G protein, 4.1G appeared to be highly concentrated underneath the
plasma membrane and specifically at cell boundaries, as protein 4.1R
and fodrin are (Fig. 7A, arrows). However, significant
accumulations of 4.1G were also observed within the cytoplasm of PC12
cells, assuming a rather punctate distribution pattern (Fig. 7A,
arrowheads), whereas any specific staining was eliminated when the
primary antibody was omitted (Fig. 7B).
-fodrin, and actin. An ~150-kDa 4.1R isoform was readily
detected in both anti-Exon16 and anti-fodrin immunoprecipitates (Fig.
8A). When the same fractions were analyzed for the presence
of fodrin, two closely migrated polypeptides of ~240 and ~235 kDa,
which correspond to
- and
-fodrin isoforms, respectively, were
detected (Fig. 8B). Moreover, when a replica blot was
examined for the presence of
-actin, an ~43-kDa immunoreactive
polypeptide was identified (Fig. 8C). In control experiments
where pre-immune serum was used in the place of primary antibodies, no
immunoreactive band was precipitated (Fig. 8, A-C).
Collectively, all these findings suggest that protein 4.1R occurs
in vivo in a supramolecular complex with fodrin tetramers and actin filaments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-fodrin
(brain spectrin) and F-actin in a series of in vitro binding
assays. We also explored the functional ability of the 4.1 homologs G-,
N-, and B-SAB domains to promote fodrin/actin co-sedimentation in
vitro. Finally, we examined the intracellular distribution of
4.1R, 4.1G, and fodrin in the neuronal PC12 cell line, and we analyzed
the potential in vivo association of 4.1R and 4.1G proteins
with fodrin by a series of co-immunoprecipitation assays.
-actin, and the
actin-associated protein tropomyosin (44). Thus, it is conceivable that
among different tissues 4.1R-10-kDa may have evolved diverse functional
activities, either behaving as a general linkage molecule that bridges
the underlying cytoskeleton to the cell membrane (e.g.
erythroid cells and brain) or assuming new roles through interactions
with major tissue-specific proteins (e.g. skeletal muscle).
A slightly enhanced fodrin/actin binding activity was observed when
both exons 14 and 15 were present even though the individual exon
appeared to suppress the binding. The biological significance of exons
14 and 15 in fodrin/actin interaction remains to be determined.
-fodrin tetramers in a series of
co-immunoprecipitation experiments. Whether 4.1R or 4.1G promotes
formation of fodrin-actin complexes more efficiently in the
physiological environment of PC12 cells is still a matter of
speculation. Nevertheless, the relative amounts of the respective
proteins available within the cell may dramatically affect the
favorable recruitment of 4.1R or 4.1G in the formation of ternary
complexes. The fact that 4.1N and 4.1B do not stimulate fodrin/actin
association, even though they are present at the boundaries of
undifferentiated PC12 cells (21, 23), is indicative of non-redundancy
among the multiple members of the 4.1 gene superfamily.
Conceivably, these proteins may be involved in the segregation and
modulation of other protein complexes associated with the plasma
membrane, in the structural organization of cell-cell contact regions
to specialized domains, or in major signal transduction pathways that
control cell growth and differentiation (49).
-fodrin, a
major component of the neuronal cytoskeleton, was established.
Consequently, the challenge of future studies will be the
identification of specific binding partners and the elucidation of the
functional activities of the multiple 4.1-proteins expressed in brain,
in order to unravel the complicated architecture of neuronal cytoskeleton.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. R. J. Bloch (University of Maryland), N. Mohandas, P. Gascard, and J. G. Conboy (University of California, Berkeley) for providing us with the fodrin and 4.1G, 4.1N, and 4.1B antibodies, respectively. M. Delannoy is also thanked for assistance with confocal microscopy (The Johns Hopkins University School of Medicine).
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FOOTNOTES |
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* This work was supported in part by Grant HL 44985 from the National Institute of Health (to E. J. B.) and by an American Heart Association grant (to S. C. H.).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.
§ Supported by a National Institutes of Health post-doctoral training grant. Current address: Dept. of Physiology, University of Maryland, Baltimore, MD 20541.
§§ To whom correspondence should be addressed: Dept. of Adult Oncology, Dana-Farber Cancer Inst., D1420B, 44 Binney St., Boston, MA 02115. Tel.: 617-632-6965; Fax: 617-632-2662; E-mail: Shu-Ching_Huang@dfci.harvard.edu.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M010581200
2 A. Kontrogianni-Konstantopoulos, E. J. Benz, Jr., and S.-C. Huang, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: SAB, spectrin/actin binding; nts, nucleotides; GST, glutathione S-transferase; PCR, polymerase chain reaction; RT, reverse transcription; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; PBS, phosphate-buffered saline; Ab, antibodies; NGF, nerve growth factor.
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