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INTRODUCTION |
The protein 4.1 family comprises a group of skeletal proteins
structurally related to the erythroid membrane skeletal protein, 4.1R,
that plays a critical role in determining the morphology and mechanical
stability of the red cell plasma membrane. These proteins are
characterized by the presence of three main conserved structural/functional domains. A 30-kDa N-terminal membrane binding domain (MBD1; also called the
FERM domain (1)), possesses binding sites for the cytoplasmic tails of
integral membrane proteins such as band 3 (2, 3), glycophorin C (4, 5),
CD44 (6), and Drosophila neurexin (7). This domain also
binds to p55 (5) and calmodulin (8), the latter interaction being
important for regulating the affinity of 4.1R-band 3 and 4.1R-CD44
interactions (6, 9). An internal 8-10-kDa domain contains the critical spectrin-actin binding activity required for membrane stability (10-13), and the C-terminal 22-24-kDa domain has recently been reported to bind the immunophilin FKBP13 (14) and NuMA (15). The
prototypical protein 4.1R has been characterized most extensively in
the erythrocyte, where it plays a critical role in maintaining the
erythrocyte's morphology and mechanical integrity. Consequently, deficiency of protein 4.1R yields an elliptocytic morphology and decreased membrane strength, leading to cellular fragmentation and
hemolytic anemia (reviewed in Ref. 16).
The complement of 4.1 proteins expressed in brain and other
nonerythroid cells is structurally more complex and functionally less
well understood than in erythroid cells. Western blot analysis suggests
that multiple protein 4.1 isoforms are expressed in most nucleated
cells (17, 18). This molecular heterogeneity is probably due not only
to post-translational modifications but also to complex alternative
pre-mRNA splicing pathways that insert or delete discrete peptides
within the polypeptide backbone (19, 20). Several of the alternative
splicing events are mediated in tissue-specific fashion, resulting in
expression of unique protein 4.1R isoforms in muscle (21, 22), brain
(20), and epithelial cells (22). Additionally, programmed changes in
alternative splicing alter the repertoire of 4.1R isoforms synthesized
during erythroid differentiation (23-25). Moreover, transcriptional
regulation of three recently discovered 4.1 genes contributes further
heterogeneity to the complement of 4.1 isoforms expressed in a given
tissue. Among these are protein 4.1G, a widely expressed homolog (14, 26); protein 4.1N, a neuronal homolog (27); and protein 4.1B, the novel
brain-enriched homolog reported here.
The brain is a particularly rich source of protein 4.1 isoforms. By
in situ hybridization analysis, 4.1R, 4.1G, 4.1N, and 4.1B
mRNAs are all expressed in distinct patterns within the brain. Several studies indicate that 4.1 protein(s) are likely to play important functional roles in the brain. For example, 4.1R has been
localized to specific neuronal populations including granule cells of
the cerebellum and dentate gyrus (28). Consistent with this
localization, behavioral studies of protein 4.1R-deficient knockout
mice have demonstrated neurological defects in fine motor coordination
and spatial learning (28). A second important 4.1 protein in brain is
4.1N, which is found in most neurons of the brain, with expression
detected at the earliest stage of postmitotic differentiation (27).
Immunofluorescence experiments show that 4.1N protein is enriched at
regions of synaptic contact between neurons, where it could potentially
play an important role in synaptic architecture and function (27). In
addition to these recent reports, earlier biochemical studies indicated
that at least one brain isoform of 4.1 possesses spectrin-actin binding activity, strongly suggesting that one function of brain 4.1 is analogous to its role in the red cell (29). However, the relationship of that biochemical activity to the newly described 4.1 genes is unknown.
Functional studies of 4.1 proteins in other nonerythroid, nonneuronal
cells indicate that selected 4.1 isoforms are imported into the
nucleus, where they may play a general structural role in nuclear
architecture (30-33) and/or may interact with splicing factors (34).
4.1 proteins have also been reported to interact with the immunophilin
FKBP13 (14), with CD44 (6), with the Drosophila protein
neurexin (7), and with pICln, a protein potentially involved in volume
regulation through association with chloride channel protein(s) (35).
This diversity of interactions suggests that 4.1 proteins play multiple
functional roles in addition to their well known function in
stabilizing the red cell membrane.
Several classes of neurons in the brain, including Purkinje cells in
the cerebellum and the majority of thalamic nuclei, appear to exhibit
little or no expression of the previously described 4.1 protein family
members (27, 28). In this paper, we present a detailed characterization
of a fourth member of the protein 4.1 family, 4.1B. This protein is
expressed in Purkinje cells and thalamic nuclei as part of a
region-specific expression profile that is distinct from that of the
other 4.1 genes in the brain. Conserved structural features of 4.1B are
presented as well as evidence that alternative splicing events govern
expression of tissue-specific 4.1B isoforms with different functional
properties. These studies suggest that 4.1B protein(s) play a critical
structural role in a subset of neurons in the brain.
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MATERIALS AND METHODS |
Isolation of 4.1B cDNA--
The dbEST data base (National
Center for Biotechnology Information, or NCBI) was screened for novel
clones with homology to 4.1R, 4.1G, and 4.1N, resulting in
identification of clone L26705. This cDNA spanned the C-terminal
coding sequences plus the 3'-untranslated region of a novel mouse
cDNA designated as protein 4.1B. Primers designed from this
sequence were used to amplify mouse 4.1B sequences (ultimately
corresponding to nucleotides 2973-4019 of mouse 4.1B cDNA,
accession number AF152247) for use as a hybridization probe. Two long
cDNAs of 3.9 and 3.5 kb, isolated from a mouse brain cDNA
library (Stratagene, La Jolla, CA), allowed us to deduce the
full-length mouse brain 4.1B primary structure. The translation initiation site was assigned based on the following criteria: it is the
first AUG in the cDNA, it is situated in a favorable Kozak
consensus sequence, and it encodes an N-terminal peptide (MTTE) that is
identical to the N-terminal sequence of 4.1G, 4.1N, and high molecular
weight isoforms of 4.1R. The mouse 4.1B gene designation is
Epb4.1l3 (36).
RNA Blot Analysis--
4.1B mRNA size was determined by
hybridization to Multiple Tissue Northern blots
(CLONTECH, Palo Alto, CA). The probe used for
Northern blot analysis of 4.1B mRNA was derived from the
3'-untranslated region of 4.1B, not overlapping the coding region. This
probe was gene-specific for 4.1B, i.e. it did not
cross-hybridize with other 4.1 mRNAs. Control blots were performed
using the ubiquitin probe supplied by the manufacturer.
In Situ Hybridization--
Experiments were carried out using
digoxigenin-labeled probes corresponding to the 3'-untranslated region
of mouse 4.1B cDNA (accession number AF152247; nucleotides
2973-4019). Fresh frozen 20 µM cryostat sections of
whole mount embryos were laid onto Superfrost Plus slides (Fisher) and
processed as described (14). Control sections were hybridized with
identical quantities of sense cRNA, and no signal was observed. The
in situ hybridization protocol has been used to discriminate
the localizations of transcripts sharing as much as 85-90% nucleic
acid identity (37).
RT-PCR Experiments--
Total RNA from mouse tissues was
prepared and transcribed into cDNA using gene-specific antisense
primers as described (19). Two µl of cDNA was amplified in a
25-µl PCR containing Taq polymerase buffer, 50 pmol each
of sense and antisense primers, 0.2 mM dNTPs, and 0.625 units of Taq polymerase. Thirty-five cycles of amplification were performed using an automated Perkin-Elmer Cetus 9700 thermal cycler under the following conditions: denaturation for 20 s at 94 °C; annealing for 20 s at 60 °C; extension for 40 s
at 72 °C. DNA fragments were analyzed by 5% polyacrylamide gel
electrophoresis. The identity of PCR products was confirmed by DNA
sequence analysis. For PCR of 4.1B SAB domain sequences, the following
primers were used to amplify mouse 4.1B sequences: sense strand,
5'-GAAGAAGACAGAAGGAAGAAGGCT-3'; antisense strand,
5'-CCACTCGTTTGTTAAGGCAG-3'. For PCR of mouse 4.1B C-terminal domain
sequences, the following primers were used: sense strand
5'-GCCCAGACGATCACGTCTGAAACCACT-3'; antisense strand, 5'-CACGGACGGATAGAAGTCAGTTGGGT-3'.
Antibodies--
The synthetic peptide N-TTKGISQTNLITTVTPEKKA-C
was used as an immunogen to raise goat anti-4.1B antibodies. This
peptide represents a portion of the unique U2 domain that is conserved
between human and mouse 4.1B but does not share sequence homology with
4.1R, 4.1G, or 4.1N. Before use on Western blots, the antibody was
affinity-purified using the immunizing peptide coupled to a Sulfolink
column (Pierce). Preimmune IgG affinity purified from the same goat was
used in control blots. Brain extracts were also probed with rabbit
polyclonal antibodies to the unique U1 domains of 4.1G (14) and 4.1N
(27). In some experiments, antibodies were preabsorbed by incubation for 2 h with a 200-fold molar excess of the appropriate 4.1 recombinant proteins expressed in the pET22b vector (Novagen, Madison,
WI) before blotting.
Mouse Tissue Preparation--
A mouse was perfused free of blood
via left ventricular/ascending aortic perfusion with PBS containing 0.5 mM di-isopropyl fluorophosphate. The brain was immediately
harvested and homogenized in ice-cold lysis buffer (100 mM
NaCl, 50 mM Trizma, pH 7.4, 2% SDS, 1% Triton X-100, 1 mM EGTA, 2 mM Pefabloc, 5 mM
benzamidine, 2 µg/ml pepstatin A, 5 µg/ml leupeptin, 5 µg/ml
aprotinin). Tissue was left on ice for 30 min with intermittent
vortexing. The tissue lysate was denatured by boiling with an equal
volume of 2× SDS denaturing buffer containing 100 mM
dithiothreitol. An aliquot of nondenatured lysate was assayed for
protein concentration using DC reagents (Bio-Rad).
Western Blot Analysis--
Detection of endogenous mouse brain
4.1 proteins was performed using fresh brain extracts isolated as
described above. 50 µg of protein was loaded per lane in a 7.5%
polyacrylamide gel. For control experiments to detect the
epitope-tagged 4.1B isoform expressed in transfected COS-7 cells,
1.5 × 106 cells were grown in two 150-mm culture
dishes for 24 h and transfected for 8 h using 30 µl of
LipofectAMINE (Life Technologies Inc., Gaithersburg, MD) and 12.5 µg
of DNA/dish. 48 h after transfection, cells were washed in PBS,
scraped off the plates in 750 µl of ice cold radioimmune
precipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% IGEPAL CA-630 (Sigma), 0.1% SDS, 2 mM Pefabloc (Roche Molecular Biochemicals), 5 mM benzamidine, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 2 µg/ml pepstatin A), and lysed for 30 min on ice with intermittent
vortexing. The supernatant was cleared of particulate materials by
centrifugation. 100 µl of protein A-agarose beads, preincubated
overnight with 10 µg of anti-hemagglutinin epitope tag antibody, was
added and allowed to bind for 6 h at 4 °C. Beads were spun down
and washed in radioimmune precipitation buffer, and associated proteins
were denatured by boiling in SDS-PAGE sample buffer and resolved by
polyacrylamide gel electrophoresis.
Proteins were transferred to a polyvinylidene difluoride membrane
(Millipore, Bedford, MA) using a semidry electroblotter (Integrated
Separation Systems Inc., Natick, MA). Nonspecific binding sites on the
membranes were blocked by incubation for 1 h at room temperature
in TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl)
containing 0.5% Tween 20, 4% nonfat dry milk, 1% BSA, and 0.02%
sodium azide. The blocking buffer also included either 2% donkey serum
(4.1G, 4.1N) or 2% rabbit serum (4.1B) to minimize subsequent
nonspecific binding of secondary antibodies. Blots were then probed
overnight at 4 °C with either anti-4.1B antibody at 0.3 µg/ml,
anti-4.1G antibody at 0.025 µg/ml, or anti-4.1N antibody at 0.025 µg/ml (all dilutions in TBS buffer). Immunoreactive bands were
visualized after incubation with secondary antibodies coupled to
horseradish peroxidase, using either a 1:3000 dilution of donkey
anti-rabbit IgG (Amersham Pharmacia Biotech) for 4.1N and 4.1G, or a
1:80,000 dilution of rabbit anti-goat IgG (Sigma) for 4.1B, together
with the Renaissance chemiluminescence detection kit (NEN Life Science Products).
Immunofluorescence of Endogenous 4.1B in Cultured PC12
Cells--
Rat PC12 cells were grown for 6 days on two-well Lab Tek
chamber slides (Nalge Nunc International, Naperville, IL) in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated horse serum, 10% heat-inactivated fetal calf serum,
and 1% penicillin/streptomycin (Life Technologies). Cells were washed
twice in PBS, fixed for 30 min at room temperature in PBS plus 4%
paraformaldehyde, and permeabilized for 10 min at room temperature in
PBS plus 0.5% Triton X-100. After an extensive wash in PBS, samples
were blocked for 1 h at room temperature in PBS plus 10 mg/ml BSA
(PBS/BSA) plus 10% goat serum. Prior to incubation with primary
antibodies, samples processed for 4.1B staining were incubated at room
temperature with an avidin-biotin blocking kit according to the
manufacturer (Vector Laboratories, Burlingame, CA). After blocking,
cells were incubated overnight at 4 °C in a humidified chamber with
PBS/BSA containing 10 µg/ml of either affinity-purified goat
anti-human 4.1B antibody or preimmune serum from the same goat. After
incubation with primary antibodies, cells were washed in PBS plus 0.1 mg/ml BSA and then incubated for 1 h at room temperature with
biotin-SP-conjugated affinity-purified donkey anti-goat IgGs (Jackson
Immunoresearch Laboratories, West Grove, PA) diluted 1:2000, followed
by fluorescein (5-((4,6-dichlorotriazin-2-yl)amino)
fluorescein)-conjugated streptavidin (Jackson Immunoresearch
Laboratories, West Grove, PA) diluted 1:15,000. After extensive washing
in PBS plus 0.1 mg/ml BSA, samples were mounted in Vectashield
containing DAPI (Vector Laboratories, Burlingame, CA) and analyzed with
a Zeiss Axiovert 135 microscope.
Measurement of Spectrin-Actin Binding Activity by Falling Ball
Viscometry--
For these assays, peptides from the human 4.1 proteins
were utilized to allow comparison with previous functional studies. Recombinant human protein 4.1R SAB bearing a C-terminal hexahistidine tag had the sequence
MEPTEAWKKKRERLDGENIYIRHSNLMLEDLDKSQEEIKKHHASISELKKNFMESVPEPRPSEWDKRLSTHSPFRTLNINGQIPTGDGRHHHHHH, corresponding approximately to the coding domains of exons 16 (single
underline) and 17 (double underline) as used in previous binding assays
(10) but now including a hexahistidine tag. The sequence of the
homologous SAB domain in human protein 4.1B was as follows:
MGNSLIKRIKGENVYVKHSNLMLEELEKTQDDLMKHQTNISELKRTFLETSTDTAVTNEWEKRLSTSPVGGRHHHHHH, including the C-terminal hexahistidine tag. The single underline indicates sequences homologous to those encoded by 4.1R exon 16, while
the double underline represents the first 45 residues encoded by the
paralog of 4.1R exon 17. This construct includes all residues with
homology to the 4.1R SAB domain defined previously. The N-terminal methionine was added to facilitate translation initiation, and the
"G" and "GGR" sequences at each end derive from restriction sites used in construction of the clone.
Recombinant proteins were purified by Cobalt column chromatography
(CLONTECH, Palo Alto, CA) using 200 mM
imidazole as the elution buffer. Spectrin was purified from human red
cells (38, 39) and nonmuscle actin derived from human platelets was
obtained from Cytoskeleton (Denver, CO). Monomeric actin was
polymerized at high concentration (5 mg/ml) to kinetically favor
formation of many short filaments, in the presence of 5 mM
Tris-HCl, pH 8.0, 50 mM KCl, 2 mM
MgCl2, 1 mM EGTA, and 1 mM ATP. The
effect of recombinant 4.1 peptides on the formation of supramolecular complex with spectrin and actin was assayed by falling ball viscometry (11, 13, 40, 41). Spectrin (1.8 µM), actin (14 µM), and various concentrations of recombinant 4.1 SAB
peptides were mixed, drawn into a 50-µl microcapillary tube, and
incubated at 4 °C for a minimum of 1 h to allow gelation. The
viscosity was correlated to the velocity of a grade 10 stainless steel
ball (0.025 inches in diameter) traveling down the microcapillary at
60° from the horizontal.
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RESULTS |
Cloning of 4.1B cDNA from Mouse Brain--
A novel mouse brain
cDNA, representing a new member of the protein 4.1 family of
skeletal proteins, was identified and isolated by a combination of
computer analysis of genetic data bases and traditional cDNA
library screening (see "Materials and Methods"). This new 4.1 family member was designated as 4.1B, reflecting its high level
expression in brain as well as its broad expression in a number of
other tissues. The 4.1B gene was mapped to mouse chromosome 17 and
assigned the gene designation Epb4.1l3 (36).
The predicted protein product of this 4.1B cDNA is 103 kDa in size.
In overall domain structure, it closely resembles 4.1G, 4.1N, and 4.1R,
including three regions of high homology to known functional domains of
protein 4.1R (Fig. 1). The most extensive conserved sequence spans residues 118-454 of 4.1B and exhibits 74%
identity to the MBD of 4.1R (also called the FERM domain due to the
homology of this region in 4.1 with ezrin, radixin, and moesin). As
defined by this phylogenetic approach, the MBD extends approximately 38 amino acids C-terminal to the domain boundary originally defined by
chymotryptic digests (42). The MBD of 4.1R interacts with integral
proteins such as band 3, glycophorin C, and CD44. While the precise
binding motifs for some of these ligands has not been defined, the
overall high sequence homology of 4.1B suggests a similar function
involving interaction with one or more integral proteins in the brain.
However, a key peptide required for 4.1R binding to band 3, the LEEDY
sequence at residues 247-251 (Fig. 1B; Ref. 43), is altered
to LEKDY in 4.1B. It therefore seems likely that the integral protein
targets for 4.1B binding may differ somewhat from those of 4.1R. It
should be noted that the membrane binding domain of these 4.1 proteins
is less homologous to ERM proteins ezrin, radixin, and moesin (~35%
amino acid identity).
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Fig. 1.
Domain organization and sequence of mouse
protein 4.1B. A, domain organization of proteins in the
4.1 family. Highly conserved domains include the MBD (72-74% identity
in pairwise comparisons), the SAB (50-70% identity), and the
C-terminal domain (CTD; ~70-80% identity). Unique
domains whose sequence is divergent among 4.1 proteins are indicated as
U1, U2, and U3. Note that the structures depicted represent composite
proteins containing all of the known alternative domains. Therefore,
since each 4.1 gene encodes multiple spliceoforms, not every translated
product contains all of the domains shown. Sources of sequences are as
follows: 4.1B, a composite protein derived from a full-length mouse
brain 4.1B cDNA (accession number AF152247) with the addition of 22 amino acids expressed only in muscle (residues 559-580 of 4.1B; see
also Fig. 4A); 4.1R, a composite protein assembled from
mouse erythroid 4.1 cDNA (L00919) with the addition of sequences
encoded by exons 14 and 15; 4.1G, mouse brain 4.1G (AF044312); 4.1N,
mouse brain 4.1N (AF061283). The structure of 80-kDa erythrocyte
protein 4.1R, which lacks the U1 and U3 domains, is shown at the
bottom for comparison. B, mouse brain protein
4.1B sequence aligned with 4.1R, 4.1G, and 4.1N (all from mouse).
Identical residues are boxed, and conserved residues are
shaded. We note that an anonymous human brain cDNA
recently deposited in GenBankTM (accession no. AB023204)
represents an apparent homolog of mouse 4.1B cDNA. This cDNA
predicts a structure similar to that of the mouse 4.1B reported here.
Another apparent homolog, DAL1 (accession no. AF069072) represents a
truncated isoform with shortened N and C termini (46).
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Another conserved domain in 4.1B corresponds to the SAB domain, whose
structure and function has been studied predominantly in mature red
blood cells. In the prototypical 4.1R, alternative splicing controls
expression of two functionally different SAB domain structures: a high
affinity isoform that binds strongly to spectrin and actin and is
abundant in red cells and a low affinity isoform that lacks a key
21-amino acid peptide and is expressed in nonerythroid cells and early
erythroid progenitors. Analysis of 4.1B expression has revealed a
similar phenomenon in which alternative splicing can generate
structurally different SAB domains. Specifically, brain 4.1B exhibits
~50% identity with the C-terminal 43 amino acids of the minimal SAB
domain of 4.1R but lacks the critical N-terminal 21-amino acid peptide.
In contrast, an isoform possessing a complete functional SAB domain is
expressed in skeletal and heart muscle (see also Figs. 4 and 7).
The C-terminal region of 4.1B constitutes a third highly conserved
domain that is found not only in the other mammalian 4.1 proteins
depicted in Fig. 1 but also in the related Drosophila 4.1 protein (44). This domain of 4.1B is highly homologous to the NuMA
binding domain of 4.1R (15) and the FKBP13 binding domain of 4.1G (14).
More specifically, 4.1B and 4.1R are approximately 93% identical over
the C-terminal 59 residues that constitute the NuMA binding domain
(4.1R residue 800 to the C terminus in Fig. 1B; Ref. 15).
Similarly, 4.1B and 4.1G exhibit 80% identity in the overlapping
C-terminal 92 residues that correspond to the FKBP13 binding domain
(4.1G residue 897 to the C terminus (14)).
In addition to these conserved domains, protein 4.1B also possesses
several unique domains whose primary sequence is not shared with other
4.1 proteins. These unique regions are designated as U1, U2, and U3
(Fig. 1A). U1 represents the N-terminal "headpiece" located upstream of the membrane binding domain in not only 4.1B but
also in the other three members of the family. 4.1B has little or no
homology to the other 4.1 proteins in this region, aside from the
initial MTTE peptide. Unique domain U2 corresponds to the "spacer"
region between the membrane binding and SAB domains. Again, this domain
is present in all four 4.1 proteins, but the primary sequence is poorly
conserved, and the function is not known. Finally, the U3 represents
another region whose position is conserved among the 4.1 proteins but
whose primary sequence is not. The amino acid content of this region in
4.1B exhibits some similarities to the large insert in the 440-kDa
isoform of human ankyrin B (45); both are highly charged with a
particular abundance of glutamate and aspartate residues and an acidic
pI, and both are highly enriched in serine and threonine residues. The
functional significance of this similarity remains to be explored.
An apparent human ortholog of the mouse 4.1B, designated as DAL1, was
recently reported (46). The respective 4.1B and DAL1 genes map to
regions of conserved synteny in human chromosome 17 and mouse
chromosome 18 (36, 46). Moreover, DAL1 protein is nearly identical in a
600-amino acid region of overlap with mouse 4.1B, extending from the
methionine at 4.1B position 118 to a point C-terminal to the
spectrin-actin binding domain (46). However, the predicted DAL1 protein
lacks the N- and C-terminal domains of mouse 4.1B, apparently due to
frameshifts in the DAL1 cDNA sequence (46).
Tissue-specific Expression of 4.1B mRNA--
Northern blot
analysis of various tissues was performed with a 4.1B cDNA probe.
As shown in Fig. 2A, the brain
exhibited high level expression of a 4.4-kb 4.1B mRNA and somewhat
lower levels of a 2.8-kb transcript. Both of these transcripts were
also observed in placenta, while kidney, heart, and lung expressed
predominantly the larger transcript. Low expression was observed in
pancreas and skeletal muscle, with no detectable 4.1B mRNA in
liver. While the 4.4-kb mRNA is consistent with the length of our
mouse brain 4.1B cDNA sequence (GenBankTM accession no.
AF152247), the structure of the 2.8-kb mRNA is less well
understood. Northern blot experiments with region-specific probes
indicated that the smaller transcript contains sequences homologous to
the coding domain for SAB and the C-terminal domain, as well as the
3'-untranslated region; however, no hybridization was observed to a MBD
probe (data not shown).
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Fig. 2.
Northern blot analysis of 4.1B mRNA.
A, human mRNA was hybridized under stringent conditions
with a 4.1B 3'-untranslated region probe. RNA size standards of 9.5, 7.5, 4.4, 2.4, and 1.35 kb are shown at the left. Sources of
RNAs are as follows: heart (lane 1), brain (lane
2), placenta (lane 3), lung
(lane 4), liver (lane 5), skeletal
muscle (lane 6), kidney (lane
7), pancreas (lane 8).
Below is shown a positive control depicting hybridization of
the same RNAs with a ubiquitin probe. B, a human master blot
(CLONTECH), containing RNAs from many individual
tissues, was hybridized under stringent conditions with a 4.1B
3'-untranslated region probe. A key showing the identity of
RNAs on the master blot is shown below.
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A more extensive survey of 4.1B mRNA expression was performed by
two additional approaches. First, a 4.1B probe was hybridized to a dot
blot containing RNA from many human tissues (Fig. 2B). High
expression was observed in many regions of the brain (rows A and B), and very high expression was observed
also in adrenal gland (coordinate D5). Little or no expression was
detected in erythropoietic tissues (bone marrow, E8; fetal
liver, G4), where 4.1R expression is highest (26). To obtain
a higher resolution of 4.1B expression patterns, we performed in
situ hybridization. Consistent with the results of the Northern
blots, Fig. 3A demonstrates that 4.1B transcripts are most abundant in brain. Lower expression was
also detected in testis, adrenal gland, and kidney. Examination of
higher resolution hybridization signals in brain for 4.1B, as well as
for 4.1R and 4.1N, revealed focal expression patterns in various
neuronal populations (Fig. 3B). In the cerebellum, 4.1B was
expressed specifically in Purkinje cells. In contrast, 4.1R and 4.1N
are expressed in the granule cell layer, with no detectable expression
in the Purkinje cells. Distinct expression patterns were also observed
in the hippocampus. Whereas 4.1B expression was restricted to pyramidal
cells of the CA1-3 region, 4.1R was localized exclusively to granule
cells of the dentate gyrus. A third pattern was observed with 4.1N,
which was expressed both in the dentate gyrus and CA1-3 regions. 4.1B
also exhibited strong expression in the thalamic nuclei, where there is
no detectable 4.1R, 4.1G, or 4.1N expression (data not shown).
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Fig. 3.
In situ hybridization of 4.1B
mRNA in mouse. A, a whole mount of a newborn mouse
was hybridized with a mouse 4.1B RNA antisense probe. At low power,
prominent expression is evident in the cerebellum (CB),
hippocampus (H), olfactory bulb (OB), olfactory
epithelium (OE), testis (T), adrenal
(A), and kidney (K). Control experiments using a
sense strand probe were completely negative (data not shown).
B, regional expression in the brain. Two areas of the brain
are shown to highlight differential expression of the 4.1 genes among
neuronal populations. Upper panel shows expression of the
indicated 4.1 RNAs in cerebellum. Whereas both 4.1N and 4.1R are
expressed exclusively in the granule cells, 4.1B is expressed
specifically in the Purkinje cells. Lower panel demonstrates
expression of 4.1 mRNAs in the hippocampus. 4.1B is expressed in
the pyramidal cells of CA1-3, while 4.1R is expressed specifically in
the granule cells of the dentate gyrus. 4.1N exhibits a broader pattern
of expression in both CA1-3 and dentate regions.
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Lower levels of 4.1B transcript were also detected in select peripheral
tissues, including the testis, adrenal gland, kidney, and
gastrointestinal tract (Fig. 3A). Examination of these
peripheral tissues at higher power revealed 4.1B expression in discrete
cell populations (data not shown): In the testis, highest expression occurs in sexually mature mice, with 4.1B transcripts predominantly localized to spermatocytes (undergoing meiosis) in the seminiferous tubules. In the adrenal gland, transcripts localize to the medullary chromaffin cells; the cellular components of the adrenal cortex are
negative. The kidney demonstrates striking regional expression, with
4.1B transcripts restricted to the convoluted tubule epithelia of the
cortex; the epithelial cells of the straight tubules, loops of Henle,
and collecting tubules in the kidney medulla are negative. In addition
to detection of high level 4.1B expression in select neurons of the
central nervous system, the enteric neurons of the gastrointestinal
tract are also positive for 4.1B transcript.
Alternative Splicing in 4.1B Pre-mRNA--
The prototypical
protein 4.1R gene utilizes alternative pre-mRNA splicing to mediate
tissue-restricted expression of several distinct 4.1R protein isoforms.
Of particular importance, the complete SAB domain is encoded by
alternative exon 16 plus constitutive exon 17, and its structure and
function is regulated by alternative splicing of exon 16 to produce
isoforms that bind with high affinity (e.g. in mature red
blood cells) or low affinity (e.g. in T lymphocytes) to
spectrin and actin. To explore whether variations in SAB structure exist among protein 4.1B isoforms, we employed RT-PCR techniques to
characterize this region of 4.1B mRNA isolated from various tissues. As shown in Fig. 4A,
this experiment yielded three distinct DNA bands in tissue-specific
patterns, indicative of regulated alternative splicing of discrete
exons in the 4.1B gene. Tissues such as kidney (lane
3) yielded only a single PCR product corresponding to a
"default" splicing pattern in 4.1B that encodes a truncated SAB
domain. The shortened SAB domain lacks a critical N-terminal peptide
and would not be expected to exhibit high affinity interactions with
spectrin. In contrast, amplification of RNA from heart (lane 2) and skeletal muscle (results not shown) generated two
products. Sequence analysis of the larger product revealed that it
encodes a complete SAB domain, including a 66-nucleotide paralog of
4.1R exon 16. The deduced primary amino acid sequences of the truncated and intact SAB domains of 4.1B are shown below the
gel in Fig. 4A. Finally, PCR analysis of brain
4.1B mRNA revealed expression of a unique isoform that includes a
novel 36-nucleotide exon, provisionally assigned as exon 15 due to its
relative position in the gene. The 12-amino acid peptide encoded by
this exon (Fig. 4A) is not homologous to any sequence of the
4.1R. Together these results strongly suggest that alternative splicing
mediates muscle-specific expression of 4.1B isoforms that can interact
with spectrin and actin, whereas 4.1B isoforms expressed in brain and
other tissues are likely to exhibit distinct function(s).
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Fig. 4.
Alternative splicing in 4.1B RNA.
A, tissue-specific alternative splicing in the SAB coding
region of 4.1B mRNA was examined by RT-PCR analysis. RNA from mouse
brain (B), heart (H), and kidney (K)
was amplified with 4.1B gene-specific primers. RT-PCR products were
analyzed by polyacrylamide gel electrophoresis followed by DNA
sequencing. Deduced structures of the products are depicted at the
left of the gel. Exons were numbered
according to the homologous exons in 4.1R and/or their relative
positions in the gene and were confirmed as independent exons by
analysis of 4.1B genomic sequences (data not shown). Note that unique
tissue-specific isoforms of 4.1B mRNA were expressed in heart and
brain. Shown below the gel are the deduced
peptides corresponding to the muscle isoform (upper
sequence), the brain isoform (middle
sequence), and tissue-nonspecific truncated SAB isoform
(lower sequence). The asterisks
indicate amino acid residues that are identical in 4.1R and 4.1B.
B, tissue-specific alternative splicing in the C-terminal
domain. Exons were numbered according to the homologous exons in 4.1R.
RT-PCR analysis was performed on RNA from brain, heart, and kidney,
using primers flanking alternative exon 21. The upper
sequence represents inclusion of the last coding peptide
that contains motifs homologous to the NuMA and FKBP13 binding sites in
other 4.1 proteins; the lower sequence indicates
the shortened isoform that lacks the C-terminal 36 amino acids.
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Alternative splicing also generates structural heterogeneity in the
C-terminal region of 4.1B mRNAs predicted to encode binding sites
for NuMA (15) and FKBP13 (14). Two classes of 4.1B-related ESTs were
found in data base searches. One class encoded the full C-terminal
domain and contains the putative binding sites for NuMA and FKBP13.
Representative clones of this class were isolated from mouse testis
(L26705) and from a human mixed fetal lung/testis/B-cell library
(AI377940 and AA928508). A second class of clones exhibited a deletion
of 117 nucleotides that encompasses the normal C terminus and normal
stop codon. Representative ESTs of this class were reported from mouse
embryo (AI466582) and bowel (AI50619) and from human fetal kidney
(AA340302) and the mixed fetal lung/testis/B-cell library (AA905681).
Clones of this type will lack 36 C-terminal amino acids and will
terminate translation at a new site in the 3'-untranslated region after
the addition of a single glutamate residue (Fig. 4B). Such
isoform(s) should be incapable of binding NuMA or FKBP13. At the
genomic level, the corresponding region of 4.1R is encoded by exon 21. Analysis of 4.1B genomic DNA sequences has revealed that this portion
of 4.1B gene structure is highly conserved, i.e. the
117-nucleotide sequence represents the paralogous exon in the 3' region
of the 4.1B gene (results not shown). However, while 4.1R exon 21 appears to be constitutively spliced, the data above indicate that its counterpart in the 4.1B gene is alternatively spliced.
To explore the tissue specificity of this alternative splicing event,
RNA from several mouse tissues was amplified with 4.1B specific
oligonucleotide primers flanking the C-terminal region. As shown in
Fig. 4B, there is considerable tissue variation in the
relative exon 21 inclusion/exclusion ratio. Brain and heart RNA
exhibited predominantly inclusion of this alternative exon, while
kidney mostly skipped this exon. This result implies that alternative
pre-mRNA splicing may regulate tissue-specific functional differences in NuMA and/or FKBP binding ability among the 4.1B isoforms
expressed in individual cell types.
Detection of 4.1B Protein Isoforms--
The existence of
alternative splicing events in the protein 4.1B transcript predicts
that multiple protein isoforms should be expressed from the 4.1B gene.
To directly investigate 4.1 protein expression, we performed immunoblot
analysis of mouse tissues using an affinity-purified anti-peptide
antibody raised against a portion of the unique U2 region of the
protein. Fig. 5 shows that prominent
immunoreactive protein doublets of 148/144 and 128/124 kDa were
expressed in mouse brain (lane 1). These 4.1B immunoreactive proteins were not observed in control experiments using
preimmune IgG (lane 2), antibody to protein 4.1N
(lane 3), or antibody to protein 4.1G
(lane 5), supporting their identity as 4.1B
specific polypeptides.
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Fig. 5.
Western blot analysis of 4.1B proteins in
brain. A, immunoblot analysis of proteins from mouse
brain was performed using anti-4.1B antibody (lane
1) or preimmune serum (lane 2). Major
4.1B polypeptides of 148/144 and 128/124 kDa were detected. Identical
aliquots of brain protein were also blotted with antibody directed
against 4.1N (lane 3) and 4.1G (lane
5). Control experiments were performed with antibodies
preabsorbed with recombinant 4.1N (lane 4) or
4.1G (lane 6). These results indicate that the
various 4.1 antibodies are gene-specific. B, immunoblot
analysis showing that the protein encoded by the cloned brain 4.1B
cDNA co-migrates with an endogenous 4.1B protein from mouse brain.
Lane 1 shows 4.1B detected in whole mouse brain
with anti-4.1B. In lane 2, COS-7 cells were
transfected with epitope-tagged 4.1B cDNA (corresponding to the
sequence shown in Fig. 1) and immunoblotted with antibody against the
hemagglutinin epitope tag. The co-migration of this band with an
endogenous brain 4.1B protein confirms that this cDNA represents an
authentic full-length 4.1B protein. Lane 3 is a
negative control in which COS-7 cells were transfected with plasmid
vector alone.
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The apparent size of the endogenous 4.1B bands in brain was larger than
predicted from the cDNA sequence. A similar observation has been
made previously for other members of the 4.1 family; 4.1R (19, 20),
4,1G (14, 26), and 4.1N (27) all exhibit apparent sizes significantly
larger than the size calculated from the known sequence. In order to
ascertain directly the apparent size of a known 4.1B protein, we
epitope-tagged the brain 4.1B isoform illustrated in Fig. 1,
transfected it into cultured cells, and detected the product via
immunoblot analysis using a tag-specific antibody. The transfected 4.1B
(Fig. 5B, lane 2) approximately co-migrated with the prominent 148-kDa endogenous band detected among
endogenous brain proteins (lane 1). As a negative
control, lane 3 shows that no protein was
detected with anti-tag antibody in cells transfected with empty vector
(lane 3).
Intracellular Localization of Protein 4.1B--
Protein 4.1R
isoforms have been detected in various intracellular locations
including the plasma membrane, the cytoplasm, and the nucleus, as well
as centrosomes and spindle poles of mitotic cells (15, 30, 31, 47, 48).
To investigate the intracellular localization of protein 4.1B, we
performed immunofluorescence microscopy on cultured PC12 cells using
affinity-purified anti-4.1B antibody. 4.1B was prominently localized to
the plasma membrane at regions of cell-cell contact (Fig.
6A). Isolated cells, in contrast, exhibited little distinct 4.1B staining (Fig. 6A,
inset). Regions of the cells corresponding to the nucleus
(Fig. 6B) did not contain any detectable 4.1B. As a control,
preimmune serum showed no plasma membrane (Fig. 6C).
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Fig. 6.
Intracellular localization of 4.1B
protein. Endogenous 4.1B in cultured PC12 cells was stained by
immunofluorescence techniques as described under "Materials and
Methods." Cells were stained with either affinity-purified goat
anti-4.1B antibody (a) or preimmune serum from the same goat
(c). Protein 4.1B specifically accumulates in areas of
cell-cell contact (a, arrow). b
represents DAPI staining of the cells shown in a.
Scale bar, 10 µm (insets of
a and b are shown at a 3 times higher
magnification).
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Spectrin-actin Binding Function of Muscle-specific 4.1B
Isoform(s)--
The studies above demonstrated that brain 4.1B
possesses a truncated SAB domain, whereas muscle isoform(s) of 4.1B
contain an intact SAB domain. To test whether the latter 4.1B protein domain retains the functional ability to interact with spectrin and
actin in ternary complexes, a falling ball viscometry assay was
performed. This assay is based on the observation that spectrin binds
to actin with low affinity in the absence of protein 4.1R and that the
addition of intact 4.1R or its SAB domain significantly enhances
spectrin-actin binding (in the context of a ternary complex that
includes 4.1R), leading to increased apparent viscosity of the
solution. Recombinant human 4.1R or 4.1B SAB domains were mixed
in vitro with spectrin and actin and then incubated to allow formation of ternary complexes. Fig. 7
shows that the prototypical SAB domain of 4.1R promotes a concentration
dependent increase in viscosity of a solution containing micromolar
concentrations of spectrin and actin. 4.1B also clearly demonstrated
the ability to promote gelation of a spectrin-actin mixture in a
concentration-dependent manner, although with reduced
efficiency compared with protein 4.1R.
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Fig. 7.
Spectrin-actin binding activity of 4.1B
protein. The ability of 4.1 proteins to form ternary complexes
with spectrin and actin was assessed using falling ball viscometry, as
described previously (10). 4.1 SAB domains were expressed in bacteria
as 8-10-kDa His-tagged peptides derived from the coding regions of
exons 16 and 17. Plots show the apparent viscosity of the solution as a
function of 4.1 peptide concentration in the presence of human
erythroid spectrin (1.8 µM) and actin filaments (14 µM). At high concentrations, the solution becomes a gel
(upper portion of plot). This assay
indicates that the muscle isoform of 4.1B possesses substantial
spectrin-actin binding activity.
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DISCUSSION |
Protein 4.1B represents a novel brain-enriched member of the 4.1 family, encoded by a unique gene on mouse chromosome 17 and human
chromosome 18 (36). 4.1B represents the fourth member of a gene family
that shares homology with the prototypical 4.1R protein in red blood
cells but exhibits distinct tissue-specific expression patterns. High
level, focal expression of 4.1B was observed in brain, particularly in
Purkinje cells of the cerebellum, pyramidal cells of regions CA1-3 of
the hippocampus, and neurons of the thalamic nuclei and olfactory bulb.
Region-specific regulation of 4.1B transcription among different
classes of neurons is thus a hallmark of 4.1B gene expression.
Nonneuronal sites of expression, including testis, adrenal, kidney, and
heart, suggest that additional transcription controls must exist in
these cell types. Regulation of 4.1B gene expression is mediated also
at the level of alternative pre-mRNA splicing. Tissue-specific
splicing events appear to control synthesis of unique 4.1B isoforms in
brain and heart/skeletal muscle; the latter isoform(s) contain a
functionally competent spectrin-actin binding domain encoded in part by
a paralog of alternative exon 16 of the 4.1R gene.
4.1B protein isoforms exhibit many of the well characterized features
of the prototypical protein 4.1R characterized earlier. The presence of
conserved functional domains suggests that 4.1B proteins function in
the skeletal architecture of specific neurons in brain in a manner
analogous to the role of 4.1R in red blood cells. In particular, the
membrane binding domain of 4.1 proteins has been shown to interact with
integral proteins such as band 3 (2, 3), glycophorin C (4, 5), CD44
(6), and Drosophila neurexin (7). The high conservation of
4.1B sequences in this region (74% identity to 4.1R over 330 amino
acids), indicates that 4.1B probably interacts with the cytoplasmic
tails of one or more integral membrane proteins in specific neurons.
Likewise, the C-terminal domain is also highly conserved, indicating
functional similarities among 4.1 proteins that may include
interactions with previously described C-terminal targets including
immunophilin FKBP13 (14) and NuMA (15). As mentioned above, a classical erythroid-type spectrin-actin binding domain is expressed only in
muscle due to the exclusion of exon 16 in other cell types. However, a
novel 12-amino acid peptide motif is substituted into this region
uniquely in brain, creating a modified domain that may bind alternative
neuronal targets, perhaps brain-specific isoform(s) of a nonerythroid
spectrin. Indeed, the existence of 4.1 homologs in brain that can bind
to spectrin has already been demonstrated (49), although the precise
relationship between those proteins and the recently cloned 4.1 homologs needs to be defined. Finally, each of the protein 4.1 genes
also encodes unique domains that are not shared with other family
members; such differences may encode novel functions not yet appreciated.
Region-specific expression of 4.1 family genes in selected neuronal
classes indicates the need for exquisite transcriptional controls
probably involving multiple promoter elements. This hypothesis is based
on in situ hybridization experiments that reveal
unique expression patterns for each of the 4.1 genes in the brain
(Refs. 14 and 27 and Fig. 3). For example, the distribution of 4.1 mRNAs in hippocampus exhibits at least three distinct expression patterns; while 4.1R is restricted to the dentate gyrus and 4.1B to the
CA1-3 region of the hippocampus, 4.1N is found in both dentate gyrus
and CA1-3. In other regions, 4.1B and 4.1N exhibited a complementary
but not mutually exclusive distribution; 4.1N is present in virtually
all neurons of the brain with the prominent exception of Purkinje cells
in the cerebellum and the majority of thalamic nuclei (27), while 4.1B
is expressed strongly in these neurons. Together these observations
suggest that transcriptional regulation of 4.1 family members in the
brain is a complex process involving multiple region-specific
promoters. A similar regulatory mechanism probably controls regional
expression among ankyrin family genes expressed in the brain (50-53).
For example, ankyrin G knockout mice, generated via a specific 5'
sequence deletion, selectively lose expression of ankyrin G in some
brain regions but not others (54). This observation indicates that
multiple promoter elements must control ankyrin expression in distinct neuronal populations.
Regulated alternative splicing is also a fundamental feature of gene
expression shared by both the 4.1 and ankyrin gene families. Protein
4.1R (20), 4.1B (this paper), and
4.1N2 all exhibit
brain-specific splicing patterns indicative of unique brain isoforms
encoded by each of these genes. Similarly, neural specific isoforms of
ankyrin G and ankyrin B have been reported (52). These results support
earlier observations that the brain is a rich source of tissue-specific
alternative pre-mRNA splicing (e.g. Refs. 55 and 56) and
suggest that the unique 4.1 isoforms generated by such splicing events
must play roles in the brain that are distinct from their functions in
nonneuronal cells.
Together, these carefully regulated transcription and alternative
splicing processes cooperate to facilitate tissue- and region-specific expression of a diverse repertoire of structural proteins in the 4.1 and ankyrin families. A subset of these proteins are likely to exhibit
neuron- and intracellular compartment-specific functions. Elucidating
the specific roles for the diverse 4.1 proteins in the brain, including
their distinct binding partners and intracellular distributions, should
provide important insights into neuronal cytoskeletal architecture and
the relevance of such structure to cell function.
§,
,
TOP
ABSTRACT
INTRODUCTION
