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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4404-4414
Characterization of Multiple Isoforms of Protein 4.1R Expressed
During Erythroid Terminal Differentiation
By
P. Gascard,
G. Lee,
L. Coulombel,
I. Auffray,
M. Lum,
M. Parra,
J.G. Conboy,
N. Mohandas, and
J.A. Chasis
From the Life Science Division, Biophysics and Biomolecular Structure
Department, Lawrence Berkeley National Laboratory, Berkeley, CA; and
the Laboratoire Hématopoïèse et Cellules Souches,
Villejuif, France.
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ABSTRACT |
In erythrocytes, 80-kD protein 4.1R regulates critical
membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4.1, generated by alternative pre-mRNA splicing, are expressed during
erythroid differentiation. Erythroid precursors use two splice acceptor
sites at the 5 end of exon 2, thereby generating two populations
of 4.1 RNA: one that includes an upstream AUG-1 in exon 2 and
encodes high molecular weight isoforms, and another that skips AUG-1 in
exon 2 and encodes 4.1 by initiation at a downstream AUG-2 in
exon 4. To begin an analysis of the complex picture of protein 4.1R
expression and function during erythropoiesis, we determined the number
and primary structure of 4.1R isoforms expressed in erythroblasts. We
used reverse-transcription polymerase chain reaction to amplify and
clone full-length coding domains from the population of 4.1R cDNA
containing AUG-1 and the population excluding AUG-1. We observed an
impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive
characterization of 4.1R primary structures in a single-cell lineage.
4.1R isoforms, transfected into COS-7 cells, distributed to the
nucleus, cytoplasm, plasma membrane, and apparent centrosome. We
confirmed previous studies showing that inclusion of exon 16 was
essential for efficient nuclear localization. Unexpectedly,
immunochemical analysis of COS-7 cells transfected with an isoform
lacking both AUG-1 and AUG-2 documented that a previously unidentified
downstream translation initiation codon located in exon 8 can regulate
expression of 4.1R. We speculate that the repertoire of primary
structure of 4.1R dictates its distinct binding partners and functions
during erythropoiesis.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE 80-kD protein 4.1R is a crucial
structural component of the plasma membrane of circulating
erythrocytes. Plasma membranes of these terminally differentiated red
cells consist of a lipid bilayer, integral proteins, and an underlying
skeletal protein latticework, which plays a critical role in supporting the bilayer and in regulating membrane properties of deformability and
mechanical strength.1,2 Well-characterized 80-kD protein 4.1R is a critical constituent of the membrane skeleton because it
participates in both horizontal associations within the skeletal protein network and in vertical associations linking the skeletal network to integral proteins embedded in the lipid
bilayer.3 The N-terminal 30-kD region of 4.1R functions as
a membrane binding domain via specific interactions with integral
proteins glycophorin C,4-9 band 3,10,11 and
CD44.12 A 10-kD domain further downstream contains the
binding sites for spectrin and actin and is essential for formation of
the ternary complex of 4.1R, spectrin, and actin, which regulates
membrane mechanical strength.13-16
However, 80-kD 4.1R represents only one member of a family of 4.1R
isoforms generated by complex alternative pre-mRNA splicing and
expressed in both nucleated erythroid precursors and nonerythroid tissues.17-21 Although mature erythrocytes express only a
restricted repertoire of 4.1R isoforms,18 we have obtained
evidence that 4.1R expression in erythroblasts is more complicated and
includes a much broader variety of 4.1R isoforms. Indeed, analysis of
4.1R mRNA structure during erythropoiesis showed that erythroid
precursors use two splice acceptor sites at the 5 end of exon 2, thereby generating two populations of 4.1R RNA: one that includes an
upstream AUG (AUG-1) and encodes high molecular weight isoforms of
4.1R, and another that skips AUG-1 and encodes 4.1R by initiation at a
downstream AUG (AUG-2) in exon 4. Initial characterization of protein
expression by Western blot analysis using antipeptide antibodies
revealed four prominent bands between ~69-135 kD.20 By
immunofluorescence microscopy, protein 4.1 epitopes localized predominantly to the plasma membrane, cytoplasm, and apparent centrosome in erythroblasts.20 Further studies in mammalian nonerythroid cells substantiate that 4.1 localizes to intracellular organelles. In centrosomes, protein 4.1 epitopes distribute along centriolar cylinders and on pericentriolar fibers.22 In
addition, 4.1 is a component of the nuclear matrix and appears to be
disbursed throughout nuclear domains involved in RNA and DNA
metabolism.23-25 Moreover, the 80-kD isoform of protein 4.1 expressed in transfected COS cells localizes within the
nucleus.26 It therefore seems likely that the various
protein 4.1 isoforms expressed in erythroblasts may contribute
significantly not only to plasma membrane structure but also to nuclear
and centrosomal architecture and function.
The spectrin-based membrane skeleton is in a dynamic state during
erythropoiesis. Kinetic studies of membrane skeletal protein synthesis
and assembly in a number of mammalian systems have led to two important
generalizations.27-31 Firstly, synthesis and assembly of
the various protein constituents are asynchronous; secondly, regulation
of assembly depends not only on the quantity of a given protein
synthesized but also on the availability of binding partners with which
it must interact for assembly. However, characterization of membrane
composition during erythropoiesis has become increasingly complicated
because it has recently become clear that a number of red cell skeletal
proteins are either products of complex alternative pre-mRNA splicing
events or are encoded by genes that are members of gene
families.21,32-38 Because the majority of studies detailing membrane protein synthesis and assembly were performed before our
knowledge of the larger number of potential players, there remain
important unanswered questions. One of these questions concerns the
expression and function of protein 4.1R since previous kinetic studies
focused on synthesis and membrane assembly of only the 80-kD
polypeptide. Furthermore, protein expression in intracellular
compartments to which 4.1 localizes, such as nucleus and centrosome,
has not been extensively characterized.
To initiate analysis of the complex picture of protein 4.1R expression
and function during erythropoiesis, we determined the number and
primary structure of 4.1R isoforms expressed in well-hemoglobinized erythroblasts. We observed a total of 18 isoforms, 9 encoded from AUG-1, 7 encoded from AUG-2, and, surprisingly, 2 lacking both of the
previously identified translation initiation sites. Of the 18 isoforms
identified, 7 isoforms comprised 87% of all of the clones
characterized, with 4 isoforms including AUG-1 and 3 isoforms excluding
AUG-1 and initiating translation at AUG-2. These 7 major isoforms
varied from one another in their patterns of inclusion and exclusion of
exons encoding the functionally critical spectrin/actin binding domain
and membrane binding domain. In transfected COS-7 cells, isoforms
distributed to the nucleus, cytoplasm, plasma membrane, and apparent
centrosome. Interestingly, individual isoforms segregated to more than
one subcellular compartment. Inclusion of exon 16 appeared to be
critical for nuclear localization, as had been previously
reported.26 Finally, the two isoforms lacking both AUG-1
and AUG-2 were expressed as proteins with translation initiated at a
previously unsuspected initiation codon located in exon 8. These two ~ 68-kD polypeptides, which included sequences encoded by exon 16, showed strong nuclear localization. We theorize that the existence of
such a diverse repertoire of 4.1R primary structure may reflect
preferential interaction of 4.1R isoforms with distinct binding
partners and varied functions of 4.1R during erythropoiesis.
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MATERIALS AND METHODS |
Erythroid progenitor isolation.
Normal human bone marrow cells were obtained after informed consent
from patients at the time of hip replacement surgery. CD34+
cells were isolated by positive selection using an immunomagnetic bead
procedure (Dynabeads M-450; Dynal, Oslo, Norway) as previously described.39 CD34+ erythroid progenitor cells
were grown in standard methylcellulose colony assays with optimal
concentrations of recombinant stem cell factor (SCF; 25 ng/mL),
recombinant interleukin-3 (IL-3; 50 U/mL), and recombinant
erythropoietin (Epo; 2 U/mL) to stimulate the proliferation of
colony-forming unit-erythroid (CFU-E) and burst-forming
unit-erythroid (BFU-E). BFU-E-derived colonies identified as
erythroid were plucked at day 11. Cells were washed twice and incubated
overnight in the presence of Epo in a modified Eagle's medium (MEM) + 20% fetal calf serum (FCS) to eliminate the rare contaminating
macrophages. The nonadherent cell suspension contained over 95%
basophilic and early polychromatophilic erythroblasts as determined by
examination of May-Grunwald-Giemsa-stained cytospin slides.
RNA preparation and DNA cloning.
Total RNA from BFU-E-derived erythroblasts was prepared as previously
described.40 Full-length 4.1R cDNAs were cloned by reverse
transcriptase-polymerase chain reaction (RT-PCR) of total RNA as
previously described.24 Primers used for PCR are shown in
Table 1 and Fig 1A. 4.1R
cDNAs including AUG-1 were amplified with a pair of primers
complementary to exon 2 and to exon 21 (Fig 1A). Amplification
of 4.1R cDNAs excluding AUG-1 was achieved by using a primer
complementary to the end of the region upstream of exon 2 and to
the beginning of exon 2, thus selecting cDNAs excluding exon 2,
in conjunction with the primer complementary to exon 21 (Fig 1A). 4.1R
cDNAs were then inserted into pSP72 vector (Promega Corp, Madison, WI).
Before cloning, the pSP72 vector was modified by insertion of a
KT3-epitope tag derived from a sequence of the C-terminal domain of the
Simian Virus 40 (SV40) large T antigen.41 Alternatively,
the KT3-epitope tag was replaced by either an HA-epitope tag from the
influenza viral hemagglutinin or a c-myc-epitope tag. cDNA clones were
screened by Southern dot blotting using 32P-labeled probes
specific for each alternatively spliced exon of 4.1R, and screening was
confirmed by DNA sequencing (Sequenase Version 2.0 DNA Sequencing Kit,
USB, Cleveland, OH). Finally, epitope-tagged 4.1R cDNAs were inserted
into the expression vector pSV2neoCMV (kindly provided by Dr P. Yaswen,
Lawrence Berkeley National Laboratory, Berkeley, CA) using an
EcoRI restriction site and used for cell transfection. In some
experiments, various ATGs throughout the 4.1R coding
sequence were mutated to CTGs using the Quikchange kit (Stratagene, La
Jolla, CA) according to the manufacturer's instructions. Mutated
clones were then processed as described above.
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| Fig 1.
Analysis of RT-PCR products of full-length 4.1R
erythroblast mRNA. Total RNA was purified from well-hemoglobinized
erythroblasts, transcribed into cDNA, and amplified by RT-PCR using
primer sets to amplify full-length coding domains from two populations
of 4.1R cDNA: those that contained AUG-1 and those that deleted AUG-1
(A). Erythroblast cDNA amplified with exon 2 (sense) and exon 21 (antisense) primers gave a product of ~2.5 kb when analyzed on a
0.7% agarose gel (B, lane 1). Eythroblast cDNA amplified with exon 2 (sense) and exon 21 (antisense) primers also gave a product of ~2.5
kb (B, lane 2). These amplification products were consistent with the
predicted sizes of protein 4.1R cDNAs either containing or deleting
AUG-1. By Southern blot analysis of amplified cDNA products, both
RT-PCR products hybridized with 80-kD full-length 4.1R DNA, identifying
them as 4.1R (C).
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Cell culture and transfections.
NIH/3T3 cells were obtained from ATCC (Rockville, MD),and COS-7 cells
were kindly provided by Dr C. Collins (UCSF, San Francisco, CA). Cells
were transiently transfected by lipofection as previously described
with minor modifications.24 Briefly, cells on coverslips were grown for 24 hours, washed with phosphate-buffered saline (PBS)
and with Opti-MEM I medium (Life Technologies Inc, Gaithersburg, MD)
and incubated for 8 to 14 hours in 1 mL of Opti-MEM I medium containing
6 µL of LipofectAMINE (Life Technologies Inc) and 2.5 µg of cDNAs encoding various epitope-tagged 4.1R isoforms. After transfection, cells were washed in PBS and incubated in growth medium
for 34 to 40 hours.
Immunofluorescence microscopy.
Samples were processed for immunofluorescence microscopy as previously
described with minor modifications.24 All steps were performed at room temperature. Forty-eight hours after transfection, cells were fixed with 3% paraformaldehyde for 20 minutes and
permeabilized with 0.5% Triton X-100 for 10 minutes. After blocking in
10% (vol/vol) goat serum for 1 hour, samples were probed with either
an anti-KT3-epitope tag monoclonal antibody (kindly provided by Dr B. Schwer, Cornell University Medical College, New York, NY) or with an
affinity purified anti-HA-epitope tag polyclonal antibody (Zymed
Laboratories Inc, South San Francisco, CA) or with a monoclonal
anti-c-myc-epitope tag antibody (Boehringer Mannheim, Indianapolis,
IN) diluted 1/10, 1/300, and 1/200, respectively. In some experiments,
samples were double stained with a monoclonal anti-HA-epitope tag
antibody (Babco, Richmond, CA) diluted 1/1,000 and a polyclonal 4.1R
antibody raised against the 21- amino acid (aa) sequence encoded by
exon 16,24 used at 10 µg/mL. After incubation with
primary antibodies, samples were incubated for 1 hour, either
with anti-mouse IgG conjugated to Texas red or anti-rabbit IgG
conjugated to fluorescein isothiocyanate (FITC; Molecular Probes Inc,
Eugene, OR), diluted 1/2,000 and 1/5,000, respectively. Coverslips were
mounted using Vectorshield containing
4,6-diamidino-2-phenylindole (DAPI) as a nuclear DNA staining
probe (Vector Laboratories, Burlingame, CA). Microscopic analysis of
the samples and image processing were performed as previously
described.24
Immunoprecipitation.
COS-7 cells, seeded at 106 in 100-mm cell culture dishes,
were grown for 24 hours, transfected as described above using 24 µL
of LipofectAMINE and 10 µg of DNA per dish and 48 hours after transfection, cells were washed twice in PBS and lyzed in 750 µL of
ice cold RIPA buffer (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1%
IGEPAL CA-630 [Sigma Chemical Co, St Louis, MO], 0.1% sodium dodecyl
sulfate (SDS), 2 mmol/L Pefabloc (Boehringer Mannheim), 2 mmol/L
benzamidine, 10 µg/mL aprotinin, 5 µg/mL leupeptin, and 2 µg/mL pepstatin A) for 30 minutes on ice. All steps were performed at
4°C. Lysate from two dishes was centrifuged and supernatant was
precleared for 1 hour with 100 µL of protein A agarose beads (Life
Technologies). Precleared supernatant was incubated overnight with 100 µL of protein A agarose beads, which had been preincubated overnight
with 10 µg of polyclonal anti-HA-epitope tag antibody. After
centrifugation, beads were extensively washed with RIPA buffer and
denatured by boiling for 10 minutes.
Western blotting.
SDS-polyacrylamide gel electrophoresis (PAGE) of samples was performed
on 6% or 7% acrylamide gels. The proteins were transferred onto
nitrocellulose membrane using a semidry electroblotter (Integrated Separation Systems Inc, Natick, MA). All steps were performed at room
temperature. After blocking for 1 hour in Tris buffered saline (TBS),
0.1% Tween-20, 4% non-fat dry milk, 1% bovine serum albumin (BSA),
0.02% sodium azide, blots were washed in TBS, 0.1% Tween-20 then
probed for 1 hour with polyclonal anti-HA-epitope tag antibody diluted
at 1µg/mL in TBS, 0.1% Tween-20, 3% BSA, 0.02% sodium azide. After
several washes, blots were incubated with anti-rabbit IgG coupled to
horseradish peroxidase (Amersham, Arlington Heights, IL) diluted at
1/1,000, as described for primary antibody, except sodium azide was
excluded. After several washes, blots were developed using the
Renaissance chemiluminescence detection kit (NEN Life Science Products,
Boston, MA).
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RESULTS |
Erythroblast RNA contains seven major protein 4.1R isoforms.
To determine the number and primary structure of 4.1R isoforms
expressed in well-hemoglobinized erythroblasts, CD 34+
cells were isolated from normal human bone marrow cells, grown in
methylcellulose with SCF, IL-3, and Epo, and BFU-E-derived colonies
plucked at day 11. Total RNA was purified, transcribed into cDNA, and
amplified by RT-PCR using primer sets to amplify full-length coding
domains from two populations of 4.1R cDNA: those that contained AUG-1
and those that deleted AUG-1 (Table 1 and Fig 1A). Erythroblast cDNA
amplification products obtained with both sets of primers were ~2.5
kilobases (kb), consistent with the predicted sizes of protein 4.1R
cDNAs either containing or deleting AUG-1 (2,569 bp and 2,552 bp,
respectively; Fig 1B). Moreover, both RT-PCR bands hybridized with
80-kD full-length 4.1R DNA by Southern blot analysis, identifying them
as 4.1R (Fig 1C). Because each PCR band presumably represented a
heterogeneous population of alternatively spliced mRNAs of similar
size, a cDNA library was constructed from each band to facilitate
characterization of individual subclones. To determine the number and
primary structure of erythroblast 4.1R isoforms, these subclones were
categorized into distinct classes using both Rsa I restriction
enzyme digestions and hybridization with exon-specific oligonucleotide
probes. Representative clones from each pattern were then completely
sequenced. A total of 18 distinct isoforms were found. Of 87 independent 4.1 cDNAs subcloned, 36 clones (41%) included AUG-1,
whereas 51 clones (59%) lacked AUG-1. Nine isoforms were encoded from
AUG-1; however, 4 isoforms comprised 87% of this set
(Fig 2). The most common form was the exon
pattern encoding the 135-kD isoform (4.1R135) previously
described in erythroblasts and nucleated nonerythroid cells.18-20,23-25 The second major pattern differed from
the first in that it excluded exon 16 (4.1R135 E16),
while the third major pattern was the 135-kD form plus exon 14 (4.1R135+E14). The final major isoform of this set was the
135-kD isoform missing exon 4 (4.1R135E4). Thus, the
significant variations among the four major isoforms encoded from AUG-1
involved inclusion or exclusion of exon 14, exon 16, encoding the
functionally critical spectrin-actin binding domain, and exon 4, encoding a region of the NH2 extension and a region of the
membrane binding domain. Seven isoforms were encoded from AUG-2 with 3 patterns dominating and comprising 89% of this group
(Fig 3A). The most common form was the exon
pattern encoding the 80-kD isoform (4.1R80),
well-characterized in mature red cells.18-20 The second
major pattern differed from the first in that it excluded exon 16 (4.1R80E16), whereas the third major pattern was the
80-kD form minus exon 5 (4.1R80E5). Therefore, in the
predominant isoforms encoded from AUG-2 the significant distinctions
among patterns involved exon 16, encoding the spectrin-actin binding
domain, and exon 5, encoding a region of the membrane binding domain.
Minor isoform patterns observed in erythroblasts varied from one
another not only in the membrane binding and spectrin/actin binding
domains but also in exons 18, 19, and 20, encoding the C-terminal
domain of 4.1R that interacts with nuclear mitotic apparatus protein
(NuMA) and elongation factor 1- .42,43
Interestingly, 2 isoforms (4.1RE2,4,19 and
4.1RE2,4,5) lacked the two exons containing, respectively, AUG-1 and AUG-2 (Fig 3B). This observation was of great surprise since
AUG-1 and AUG-2 are, to date, the only documented active translation
initiation sites.
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| Fig 2.
Diagram of protein 4.1R isoforms produced by initiation
of translation at AUG-1. At the top is pictured 4.1R mRNA depicting the
alternatively spliced exons, constitutive exons, and noncoding exons.
The two translation initiation sites, AUG-1 and AUG-2, are in exon
2 and exon 4, respectively. The nine distinct protein 4.1R
isoforms produced by initiation of translation at AUG-1 are depicted
below, along with the percentage that each splice variant represented
of the total clones analyzed.
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| Fig 3.
Diagram of protein 4.1R isoforms lacking AUG-1 and
protein 4.1R isoforms lacking AUG-1 and AUG-2. At the top is pictured
4.1R mRNA depicting the alternatively spliced exons, constitutive
exons, and noncoding exons. The two translation initiation sites, AUG-1
and AUG-2, are in exon 2 and exon 4, respectively. (A) The seven
distinct protein 4.1R isoforms produced by initiation of translation at
AUG-2 are shown, along with the percentage that each splice variant
represented of the total clones analyzed. (B) The two 4.1R isoforms
that lacked both exon 2 and exon 4, the exons containing,
respectively, AUG-1 and AUG-2.
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Major 4.1R isoforms are expressed in transfected COS-7 cells.
To characterize the proteins encoded by the seven major 4.1R mRNA
isoforms expressed in erythroblasts, we transfected COS-7 cells with
HA-epitope tagged constructs corresponding to each isoform.
Immunoprecipitates of transfected cell lysates were analyzed by Western
blots probed with a polyclonal antibody specific for the HA-epitope tag
(Fig 4). 4.1R135,
the predominant isoform translated from AUG-1, migrated at ~136 kD
(Fig 4A; lane 2), a molecular weight very close to the 135 kD
previously reported for this isoform.18-20 The deletion of
exon 16 resulted in a slight decrease in protein size with
4.1R135E16 migrating as a protein of ~131 kD (Fig 4A;
lane 3). A third isoform, similar to 4.1R135 but including
exon 14 (4.1R135+E14), showed a molecular weight of ~138
kD (Fig 4A; lane 4), whereas a fourth isoform missing exon 4 (4.1R135E4) was expressed as a protein of ~116 kD (Fig
4A; lane 5). In parallel experiments, the three major isoforms
translated from AUG-2 were analyzed. The predominant isoform of this
group, 4.1R80, showed an expected size of ~79 kD (Fig 4B;
lane 2). Deletion of exon 16 (4.1R80E16) or of exon 5 (4.1R80E5) resulted in a small decrease in protein size,
both polypeptides migrating at ~77 kD (Fig 4B; lanes 3 and 4). The
expression of each of these proteins was specific because
immunoprecipitates of lysates of cells transfected with the empty
expression vector showed no expressed protein (Fig 4A and B; lane 1).
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| Fig 4.
Expression of the seven major protein 4.1R isoforms in
transfected COS-7 cells. The major 4.1R isoforms identified by RT-PCR
of erythroblast total RNA were expressed as HA-epitope-tagged proteins
in transfected COS-7 cells. Proteins were immunoprecipitated from
precleared cell lysates using a polyclonal anti-HA-epitope tag
antibody and analyzed by SDS-PAGE on a 6% (A) or 7% (B) acrylamide
gel. (A) Cells transfected with empty expression vector (lane 1);
HA-tagged 4.1R135 (lane 2); HA-tagged
4.1R135 E16 (lane 3); HA-tagged 4.1R135+E14
(lane 4); and HA-tagged 4.1R135E4 (lane 5). (B) Cells
transfected with empty expression vector (lane 1); HA-tagged
4.1R80 (lane 2); HA-tagged 4.1R80E16 (lane
3); HA-tagged 4.1R80E5 (lane 4). HA-tagged 4.1R isoforms
( ). IgG heavy chains migrating ~55 kD (+). Fainter bands
migrating between the HA-epitope-tagged proteins and IgG heavy chains
are likely to represent proteolytic fragments of the epitope tagged
proteins (see Results). Note that 4.1R80E5 is expressed
at a much lower level than the other 4.1R isoforms.
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Of note, several bands migrating between the 4.1R isoforms and the IgG
heavy chains were present on the Western blot analysis (Fig 4A and B).
These bands were likely comprised of proteolytic fragments of the
expressed 4.1R isoforms because they were not present in cells
transfected with empty vector. However, another possibility was that
these additional bands might represent smaller 4.1R isoforms translated
from alternative AUGs located downstream of the first translation
initiation codon in the transfected cDNA. Such a phenomenon has been
reported for the generation of both glycophorin C and glycophorin D
from glycophorin C mRNA.44 To investigate this hypothesis,
we mutated the two in-frame AUGs downstream of AUG-1 in
4.1R135, one located in exon 4 and the other in exon 8. In
addition, in 4.1R80 we mutated the AUG in exon 8, which
represents the first initiation codon downstream of AUG-2, the normal
site of translation initiation in 4.1R80. Western blot
analysis of immunoprecipitates of lysates of cells transfected with the
mutant cDNAs showed that the AUG  CUG (Met Leu)
mutations did not alter the pattern of polypeptide bands (data not
shown). These observations strongly argue against the possibility that
the transfected cells expressed several 4.1R isoforms derived from the
unique 4.1R cDNA used for transfection and confirmed that the
additional bands observed on the Western blots were indeed proteolytic
fragments of the expressed proteins.
4.1R isoforms distribute broadly within the cell.
To test the hypothesis that individual 4.1R isoforms segregate to
distinct subcellular compartments, we studied the distribution pattern
of the seven major isoforms in transfected COS-7 cells by
immunofluorescence microscopy. We observed that 4.1R135
localized to the cytoplasm and to a similar extent to the nucleus with
exclusion of the nucleoli
(Fig 5a through c). In
contrast, 4.1R135E16 was predominantly expressed in the
cytoplasm with no staining of the nucleus (Fig 5d through f).
This result suggested that exon 16 encoded a key determinant for
nuclear localization. Cells transfected with both isoforms also showed
an accumulation of protein in a distribution evoking membrane ruffles
that appeared as bright regions located at the periphery of
the cell (particularly obvious in the cells displayed in Fig 5b and
f).
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| Fig 5.
Expression of HA-epitope-tagged
4.1R135 and 4.1R135E16 isoforms in
transfected COS-7 cells. COS-7 cells were transfected with
HA-epitope-tagged 4.1R135 or 4.1R135E16
isoforms, fixed with 3% paraformaldehyde, permeabilized with 0.5%
Triton X-100, and processed for immunofluorescence using a polyclonal
anti-HA-epitope tag antibody as primary antibody and anti-rabbit IgG
coupled to FITC as secondary antibody. Three fields of cells
transfected with each 4.1R isoform are displayed. 4.1R135
localized to the cytoplasm and to a lesser extent to the nucleus with
exclusion of the nucleoli (a through c). Because the intensity of
nuclear and cytoplasmic staining is very similar in cells transfected
with 4.1R135, we highlighted the nucleus of the cell shown
in (c) by showing DAPI staining superimposed with the FITC staining
(see inset in lower left corner of [c]). In contrast,
4.1R135E16 was exclusively expressed in the cytoplasm
with no staining of the nucleus (d through f). Scale bar: 10 µm.
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Similar observations were made in COS-7 cells cotransfected with
4.1R80 and 4.1R80E16, two distinct 80-kD
4.1R isoforms either containing or lacking exon 16, respectively. To
allow independent visualization of these isoforms, when coexpressed in
the same cell, 4.1R80 was tagged with the c-myc-epitope
tag while 4.1R80E16 was tagged with the HA-epitope tag.
As shown in Fig 6, c-myc-epitope tagged
4.1R80 was strongly expressed in the nucleus as well as in
the cytoplasm and membrane ruffles (Fig 6a). In marked contrast,
HA-epitope tagged 4.1R80E16 localized poorly to the
nucleus but was expressed in the cytoplasm as well as in membrane
ruffles (Fig 6b). Superimposition of these images confirmed the
distinct nuclear distribution of these two isoforms (Fig 6c). Thus,
deletion of exon 16 resulted in a dramatic decrease in nuclear
expression of both 4.1R80 and
4.1R135.
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| Fig 6.
Coexpression of c-myc-epitope-tagged 4.1R80
and HA-epitope-tagged 4.1R80E16 isoforms in transfected
COS-7 cells. COS-7 cells were cotransfected with c-myc-epitope-tagged
4.1R80 and HA-epitope-tagged 4.1R80E16
isoforms, fixed with 3% paraformaldehyde, permeabilized with 0.5%
Triton X-100, and processed for immunofluorescence using a monoclonal
anti-c-myc-epitope tag antibody and a polyclonal anti-HA-epitope tag
antibody as primary antibodies and anti-mouse IgG coupled to Texas red
and rabbit IgG coupled to FITC as secondary antibodies. A cell probed
with antibody to the c-myc-epitope tag showed that 4.1R80
was strongly expressed in the nucleus as well as in the cytoplasm and
membrane ruffles (a). In marked contrast, the same cell probed with
antibody to the HA-epitope tag (b) showed that 4.1R80E16
localized poorly to the nucleus but was strongly expressed in the
cytoplasm as well as in membrane ruffles. Superimposed images of (a)
and (b) are shown in (c). Scale bar: 10 µm.
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In addition, we consistently observed stronger nuclear staining with
4.1R80 than with 4.1R135 (compare Fig 5a
through c with Fig 6a). Furthermore, the percentage of cells showing
nuclear staining was higher in cells transfected with
4.1R80 than in cells transfected with 4.1R135
(90% and 60%, respectively). These observations suggest that the
presence of the N-terminal extension, encoded when translation is
initiated at AUG-1, impacts the extent of 4.1R expression in the
nucleus.
Considered in aggregate, these data suggest that individual 4.1R
isoforms can be expressed in various cell compartments.
A single 4.1R isoform can localize to more than one subcellular
compartment.
Because conventional microscopy cannot decisively discriminate
cytoplasmic from nuclear localization or plasma membrane from cytoplasmic expression, we analyzed transfected cells by confocal immunofluorescence microscopy. Analysis of sequential sections of a 3T3
cell transfected with a KT3-epitope tagged 4.1R80 construct
confirmed that the 4.1R80 isoform was strongly expressed in
the nucleus (Fig 7), as had been observed
in transfected COS-7 cells. In addition, these sections showed clearly
that the protein was also expressed in the plasma membrane, which
appeared as a bright rim outlining the contour of the cell. Finally,
several of these sections revealed two bright immunofluorescent spots
located in the cytoplasm on either side of the nucleus. The size of
these spots and their position in relation to the nucleus and to one
another in an interphase cell suggest that they represent centrosomal
expression of 4.1R80. This observation is consistent with
our prior data that 4.1 antipeptide antibodies localize to the
pericentriolar matrix of the centrosome.20,22 Hence,
confocal microscopy confirmed our surprising findings that a single
4.1R isoform can localize to more than one subcellular compartment.
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| Fig 7.
Confocal z-sections of a NIH/3T3 cell transfected with
KT3-epitope-tagged 4.1R80. NIH/3T3 cells were transfected
with KT3-epitope-tagged 4.1-R80, fixed with 3%
paraformaldehyde, permeabilized with 0.5% Triton X-100, and processed
for immunofluorescence using a monoclonal anti-KT3-epitope tag
antibody as a primary antibody and anti-mouse IgG coupled to Texas red
as a secondary antibody. Cells were analyzed by confocal microscopy.
The 4.1R80 isoform was strongly expressed in the nucleus,
the plasma membrane, and apparent centrosomes. Every other z-section of
a series of 18 0.5-µm z-sections are displayed. Scale bar: 10 µm.
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4.1R isoforms lacking both AUG-1 and AUG-2 can still be expressed.
Two of the eighteen 4.1R mRNA isoforms identified by RT-PCR of
erythroblast total RNA lacked the AUGs encoded by exon 2 and exon 4. Because these AUGs represent the only two translation initiation codons thought to be active in 4.1R expression, the presence
of isoforms lacking both AUG-1 and AUG-2 was totally unexpected. To
determine whether an unsuspected downstream initiation codon might be
functional, COS-7 cells were transfected with one of these two
isoforms, HA-epitope tagged 4.1RE2,4,5 (Fig 8A [see page
4410]), and analyzed immunohistochemically. In immunoprecipitates of
lysates of transfected COS-7 cells the HA-epitope-tagged protein migrated as an ~68-kD protein (Fig 8B). Transfected
cells were then analyzed by double-label immunofluorescence microscopy
using a monoclonal HA-epitope tag antibody and a polyclonal antibody raised against the 21 amino acids of exon 16 of 4.1R (Fig 8C). Probing
with the HA-epitope tag antibody (Fig 8C, panel a) showed very
strong nuclear expression of this ~68-kD isoform with exclusion of
the nucleoli, and weak cytoplasmic staining. An identical pattern of
staining was obtained with the 4.1R antipeptide antibody (Fig 8C; panel
b), confirming that the expressed protein was, indeed, a 4.1R isoform.
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| Fig 8.
Expression of HA-epitope-tagged 4.1RE2,4,5
isoform in transfected COS-7 cells. COS-7 cells were transfected with
HA-epitope-tagged 4.1R80E2,4,5 isoform (exon map
shown in [A]). (B) Protein was immunoprecipitated from precleared
cell lysates using a polyclonal anti-HA-epitope tag antibody and
analyzed by SDS-PAGE on a 7% acrylamide gel. The HA-epitope-tagged
protein migrated at ~68 kD. Lane 1, cells transfected with empty
expression vector; lane 2, cells transfected with HA-tagged
4.1-R80E2,4,5. HA-tagged 4.1R isoforms (). IgG
heavy chains migrating ~55 kD (+). (C) In other experiments,
transfected cells were analyzed by immunofluorescence microscopy. Cells
were fixed with 3% paraformaldehyde, permeabilized with 0.5% Triton
X-100, and double-stained using a monoclonal anti-HA-epitope tag
antibody and a polyclonal anti-4.1R antibody raised against the 21 amino acids of exon 16 as primary antibodies and anti-mouse IgG coupled
to Texas red and anti-rabbit IgG coupled to FITC as secondary
antibodies. Cells, reacting with the HA-epitope tag antibody, showed
very strong nuclear expression of 4.1RE2,4,5 with exclusion
of the nucleoli and weak cytoplasmic staining (a). An identical pattern
of staining was obtained with the anti-4.1R peptide antibody (b).
Superimposed images of (a) and (b) are shown in (c). Scale bar:
10µm.
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Analysis of the 4.1R sequence revealed a candidate translation
initiation site located at the end of exon 8 (Fig 9). A number of observations argued in
favor of its being the relevant codon used to express the ~68-kD
protein from 4.1RE2,4,5. This particular AUG was the first
AUG downstream of exon 4 that was in frame with the 4.1R amino acid
sequence. In addition, it was surrounded by an optimal consensus Kozak
sequence (GTCATGG) required for proper translation
initiation.45 Utilization of this codon for translation initiation would result in a polypeptide of ~65 kD, thus
corresponding closely to the observed molecular weight of expressed
4.1RE2,4,5 protein. Significantly, when this AUG was mutated
to CUG (Met Leu), the ~68 kD polypeptide was no
longer detectable by Western blot analysis of immunoprecipitates of
lysates of cells transfected with the mutant cDNA (data not shown). We,
therefore, speculate that in the absence of the two previously
characterized translation initiation codons, a third AUG located in
exon 8 can effectively initiate translation of smaller 4.1R isoforms.
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| Fig 9.
Schematic diagram of exon 8 of protein 4.1R. Amino acid
sequence at the 3 end of exon 8 consisting of an AUG
codon surrounded by an optimal consensus Kozak sequence with a purine
(G) in position  3 and a G in position +4. This is the first AUG
codon downstream of AUG-2, which is in frame with the 4.1R coding
sequence, therefore making it a good candidate codon for initiation of
translation of the two isoforms lacking both AUG-1 and AUG-2.
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DISCUSSION |
A major finding of the current study is that the well-hemoglobinized
erythroblast expresses an impressive repertoire of 4.1R isoforms, which
includes 7 major and 11 minor splice variants. Although prior reports
have suggested the presence of more than a single 4.1R isoform in
various cells and tissues,18-21;24;46-52 the current
studies provide the first definitive characterization of the array of
4.1R primary structures in a single-cell lineage. Interestingly, the
predominant splicing event between the major isoforms initiated at both
AUG-1 and AUG-2 involved inclusion or exclusion of exon 16. This
particular exon, along with exon 17, encodes key residues for efficient
interaction of 4.1R with spectrin and actin.13 In the
mature erythrocyte, formation of the spectrin/actin/4.1 ternary complex
is critical for normal membrane mechanical
strength.14-16,53 Our current data emphasize that
expression of exon 16 could also play an important functional role in
erythroblasts.
Also striking was the finding that transfected protein 4.1R isoforms
show a very broad distribution within the cell with localization to the
plasma membrane, membrane ruffles, cytoplasm, centrosomes, and nucleus.
These results confirm earlier immunofluorescent observations showing
endogenous 4.1 in similar subcellular
compartments.22-24,26,47,50 However, because antibodies to
4.1R can recognize more than one isoform, it has previously been
impossible to track either the distribution of a single isoform or to
determine the presence of more than one 4.1R isoform within the same
subcellular compartment. Of great surprise to us was the observation
that a single isoform can distribute to multiple sites. Indeed, the
confocal microscopic analysis of 3T3 cells transfected with the
KT3-epitope-tagged 4.1R80 construct showed conclusively
that 4.1R80 was expressed in the nucleus, plasma membrane,
and apparent centrosome. Earlier this year, Luque and colleagues
reported that 4.1R80 expressed in transfected COS cells
distributed in most cells within the nucleus and along a network of
cytoplasmic filaments and that a smaller population of cells showed
overexpression of the protein within the cytoplasm.26
Although we cannot completely rule out the possibility that
overexpression might have altered the pattern of localization that we
observed, it is important to note that localization patterns in the
plasma membrane, the nucleus, and the centrosome similar to those
described here for epitope-tagged 4.1R in transfected cells have
definitely been observed for endogenous protein 4.1. We speculate that
diverse posttranslational modifications of the 4.1R80
isoform may contribute to the differential subcellular sorting.
Several exons appeared to influence 4.1R localization in the nucleus.
Clearly, exclusion of exon 16 resulted in a dramatic decrease in
nuclear expression of the protein. This was true for the 80-kD isoform
translated from AUG-2, as previously reported by Luque et
al,26 but also for higher molecular weight isoforms translated from AUG-1. We conclude that a motif present in exon 16 performs a critical role in nuclear targeting, as we and others have
previously suggested.26,54 Such a motif (KKKRER) has been shown to be necessary but not sufficient for nuclear targeting of
4.1R.26 In addition, we observed that nuclear expression of
the AUG-1 isoform 4.1R135, containing exon 16, was lower
than that of its AUG-2 counterpart, 4.1R80. These results
strongly suggest that the 209 amino acid N-terminal extension has a
negative impact on nuclear localization of the polypeptide. It is
unlikely that this effect results solely from the increase in protein
size because even larger proteins have been shown to be efficiently
transported to the nucleus.55,56 A more probable
explanation is that expression of the N-terminal extension produces an
overall protein conformation that either masks recognition of nuclear
localization sequence(s) or inhibits binding of 4.1R to a protein
involved in nuclear import of polypeptides.
A number of different 4.1R isoforms localized to the plasma membrane of
transfected cells. Interestingly, this was observed even for isoforms
lacking a fully functional spectrin/actin binding domain (ie, lacking
exon 16). These data emphasize that in nucleated cells, sequence motifs
in addition to those encoded by exon 16 can regulate assembly of 4.1R
onto the plasma membrane. We were also interested to observe an
accumulation of protein 4.1R in structures evoking membrane ruffles,
regions of the plasma membrane overlying dynamically reorganizing
bundles of actin-containing microfilaments. These specialized
subdomains of the plasma membrane have been described, for example, at
leading lamellae of migrating cells,57,58 sites of
injury,59 or following cell activation by various growth
factors.60-65 It is pertinent that Bessis has previously
described membrane motility in developing erythroblasts.66 Membrane ruffles are enriched in the actin-associated proteins, alpha-actinin, and fimbrin.62 Of particular note, the
4.1R80 binding partner, spectrin, has also been identified
in these structures,60,62 and it has been proposed that
spectrin links growth factor receptors to actin microfilaments of
ruffling membranes.60 In nucleated cells, protein 4.1R may
anchor the spectrin network to membrane receptors in a manner analogous
to the way it anchors the spectrin-actin network to glycophorin C, band
3 (and possibly CD44) in the erythrocyte plasma membrane. Ruffling in
nucleated cells appears to be regulated by both protein
phosphorylation61,62,64-67 and calmodulin
binding.67 Because 4.1R is a substrate for various protein
kinases68 and its interactions with the plasma membrane are
tightly regulated by calmodulin,11,12,69 it is tempting to
speculate that protein 4.1R may participate in the dynamic interaction
of actin microfilaments with various components of the plasma membrane
in membrane ruffles.
The 30-kD plasma membrane binding domain, encoded by exons 4 through
12, has been shown to mediate the interaction of 4.1R with
transmembrane proteins glycophorin C, band 3, and
CD44.4-7,10-12 Exon 5, one of the alternatively spliced
exons within this domain, contains the LEEDY sequence implicated in
4.1R-band 3 interaction.70 In the current study, we showed
that the 4.1R80E5 isoform was expressed poorly when
transfected into COS-7 cells. This was also true for another isoform
lacking exon 5, 4.1R80E5,16,18 (data not shown).
Moreover, both proteins accumulated in cytoplasmic inclusion bodies in
a large proportion of transfected cells (unpublished data). We
hypothesize that deletion of exon 5 may result in an overall misfolding
of the protein, thus altering its processing and targeting to the
plasma membrane. Alternatively, the absence of binding sequences, such
as the LEEDY motif, may impair either sorting of the protein to the
plasma membrane or assembly onto the membrane.
The two previously defined translation initiation codons responsible
for 4.1R expression are located within exon 2 and exon 4. Identification of two isoforms (4.1RE2,4,5 and
4.1RE2,4,19) lacking both of these translation initiation
codons was, therefore, unexpected. However, our immunofluorescence
observations in COS-7 cells transfected with 4.1RE2,4,5
documented that an additional translation initiation codon can
potentially regulate expression of 4.1R. Immunoprecipitates of lysates
of transfected COS-7 cells confirmed the expression of a 68-kD 4.1R
polypeptide. These data clearly show that a formerly unsuspected
downstream initiation codon can be functional. Analysis of the 4.1R
sequence reveals the presence of a candidate AUG located at the end of
exon 8. This is the first AUG codon downstream of AUG-2, which is in
frame with the 4.1R coding sequence and which is surrounded by an
optimal consensus Kozak sequence with a purine in position 3 and
a G in position +4 (GTCATGG).45 Of note, the use
of this codon for translation initiation is in accordance with the
observed molecular weight of 4.1RE2,4,5 expressed in
transfected COS-7 cells, and mutation of this codon results in the
disappearance of the protein. We have previously described a protein of
~69 kD on Western blots of human erythroblasts, further suggesting
the possibility that isoforms lacking both AUG-1 and AUG-2 may be
physiologically relevant.
The terminally differentiating erythroblast and the circulating mature
erythrocyte differ dramatically from one another both structurally and
functionally. The principal functions of the erythroblast are to
synthesize hemoglobin and, by the conclusion of terminal
differentiation, to have assembled a mechanically strong yet deformable
plasma membrane. To perform these functions an erythroblast possesses
the subcellular organelles and biochemical machinery essential for
protein synthesis and mitosis. To import the iron essential for
hemoglobin synthesis, the erythroblast plasma membrane undergoes
repeated receptor-mediated endocytosis.71 Plasma membrane
vesiculation, in the form of exocytosis, also plays a crucial role in
remodeling of the reticulocyte membrane.72-74 On the other
hand, the primary function of the terminally differentiated erythrocyte
is to efficiently deliver oxygen. In dramatic contrast to the
erythroblast plasma membrane, the plasma membrane of the mature
erythrocyte undergoes neither endocytosis nor exocytosis. Indeed,
vesicle formation is extremely detrimental to the erythrocyte, resulting in loss of surface area and loss of normal membrane deformability.75 These marked differences in the cellular
functions of the erythroblast and erythrocyte are reflected in
substantial differences in their subcellular biochemistry. We report
here that one of these biochemical differences is clearly protein 4.1R isoform expression. Although a single 4.1R isoform, 4.1R80,
is the predominant form of 4.1R in erythrocytes, erythroblasts express
additional 4.1R isoforms with functional activities potentially relevant to microtubule nucleation, nuclear function, and plasma membrane vesiculation. We speculate that the repertoire of primary structure of 4.1R described in this report dictates distinct binding partners and functions of 4.1R during erythropoiesis.
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ACKNOWLEDGMENT |
We thank Dr S.W. Krauss (LBNL, Berkeley, CA) for helpful discussions.
We also thank Dr B. Schwer (Cornell University Medical College, New
York, NY) for providing us with the anti-KT3-epitope tag antibody and
Dr C. Collins (UCSF, San Francisco, CA) for providing us with COS-7
cells. We are very grateful to Drs D. Callahan and K. Benson (LBNL,
Berkeley, CA) for their invaluable help in cell imaging and to Derek
Clark for his help in figure processing.
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FOOTNOTES |
Submitted April 21, 1998;
accepted July 27, 1998.
Supported by National Institutes of Health Grants DK26263 and DK32094
and by the Director, Office of Health and Environme |
Top
Abstract
Introduction