Download (beta)II spectrins - Journal of Cell Science

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Journal of Cell Science 113, 2023-2034 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS0962
Identification of a novel C-terminal variant of βII spectrin: two isoforms of βII
spectrin have distinct intracellular locations and activities
Nandini V. L. Hayes1, Catherine Scott1, Egidius Heerkens2, Vasken Ohanian2, Alison M. Maggs3,
Jennifer C. Pinder3, Ekaterini Kordeli4 and Anthony J. Baines1,*
1Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, England
2Department of Medicine, University of Manchester, Oxford Road, Manchester, M13 9WL, England
3MRC Muscle and Cell Motility Unit, The Randall Institute, King’s College London, 26-29 Drury Lane, London
4Institut J. Monod, CNRS, Universite Paris 7, Tour 43, 2 place Jussieu, 75251 Paris cedex 05, France
WC2B 5RL, England
*Author for correspondence (e-mail: [email protected])
Accepted 20 March; published on WWW 10 May 2000
SUMMARY
It is established that variations in the structure and
activities of βI spectrin are mediated by differential mRNA
splicing. The two βI spectrin splice forms so far identified
have either long or short C-terminal regions. Are analogous
mechanisms likely to mediate regulation of βII spectrins?
Thus far, only a long form of βII spectrin is reported in the
literature. Five human expressed sequence tags indicated
the existence of a short splice variant of βII spectrin. The
occurrence and DNA sequence of the short C-terminal
variant was confirmed by analysis of human and rat cDNA.
The novel variant lacks a pleckstrin homology domain, and
has 28 C-terminal residues not present in the previously
recognized longer form. Transcripts of the short C-terminal
variant (7.5 and 7.0 kb) were most abundant in tissues
originating from muscle and nervous system. Antibodies
raised to a unique sequence of short C-terminal variant
recognized 240 kDa polypeptides in cardiac and skeletal
muscle and in nervous tissue; in cerebellum and forebrain,
additional 270 kDa polypeptides were detected. In rat heart
INTRODUCTION
Spectrin is a cytoskeletal protein essential for the determination
of cell shape, the resilience of membranes to mechanical stress,
the positioning of particular transmembrane proteins within the
plane of a membrane, and the organization of organelles and
molecular traffic (reviewed in e.g. Bennett and Gilligan, 1993;
De Matteis and Morrow, 1998; Holleran and Holzbaur, 1998).
These functions require interactions with a wide variety of
protein and lipid ligands, among which are actin and diverse
integral and peripheral membrane proteins. It is becoming
increasingly clear that functions of spectrin in different cell
types or cellular compartments are mediated by isotypes of
spectrin that are the products of several spectrin genes, or that
vary structurally though differential mRNA splicing.
Spectrin is tetramer of two α and two β chains (for a review
of spectrin structure, see Viel and Branton, 1996). The α and
and skeletal muscle, both long and short C-terminal forms
of βII spectrin localized in the region of the Z line. The
central region of the sarcomere, coincident with the M line,
was selectively labeled with antibodies to the short Cterminal form. In cerebellum, the short form was not
detectable in parallel fibers, structures in which the long
form was readily detected. In cultured cerebellar granule
neurons, the long form was dominant in neurites, with the
short form being most abundant in cell bodies. In vitro, the
short form was found to lack the binding activity for the
axonal protein fodaxin, which characterizes the C-terminal
region of the long form. Subcellular fractionation of brain
revealed that the short form was scarcely detectable in postsynaptic density preparations, in which the long form was
readily detected. We conclude that variation in the structure
of the C-terminal regions of βII spectrin isoforms correlates
with their differential intracellular targeting.
Key words: Spectrin, Muscle, Heart, Neuron
β chains lie loosely twined side by side to form anti-parallel
dimers that self-associate to give the tetramer. Currently two
mammalian genes encoding α spectrin polypeptides (α1 and
αII) are known, as are four genes for β spectrins (sptb1
encoding βI spectrin, sptb2 encoding βII, sptbn2 encoding βIII
and elf) (Hu et al., 1992; McMahon et al., 1987; Mishra et al.,
1998; Ohara et al., 1998; Sahr et al., 1990; Stankewich et al.,
1998; Winkelmann et al., 1988). The archetypal spectrin, the
form found in red blood cells, is composed of αI and βI
polypeptides.
The β chains are of great interest since they contain most of
the binding activities of the spectrins. The N-terminal region
contains tandem calponin homology domains that bind F-actin
(Carugo et al., 1997). Most of the length of the chain is made
up of several approx. 106-residue repeats that form triple
helices, the most C-terminal of which is a partial repeat that is
a site for association with the α chain (see Viel and Branton,
2024 N. V. L. Hayes and others
1996). The C-terminal region after the last (partial) helical
repeat is a source of structural variation between β spectrins.
The archetypal red cell βI spectrin has a short C-terminal
region of 52 amino acid residues. When βI gene products were
molecularly cloned from muscle, a different C-terminal region
was detected (Winkelmann et al., 1990a). This form was longer
(about 230 residues). To distinguish the long and short forms,
Winkelmann and Forget (1993) proposed that the forms should
be given subtype (Σ) designations in the order of their
discovery: thus the short form is βIΣ1; the long form is βIΣ2.
The long C-terminal region contains within it a pleckstrin
homology (PH) domain (Musacchio et al., 1993). Cloning of
βII and βIII spectrins revealed similar C termini that included
PH domains (Hu et al., 1992; Ohara et al., 1998; Stankewich
et al., 1998). PH domains are present in a variety of proteins
involved in signal transduction, as well as cytoskeletal proteins
(Rebecchi and Scarlata, 1998). The interaction of the PH
domain with inositol phospholipids seems to regulate some of
spectrin’s membrane interactions (Godi et al., 1998).
About 100 amino acid residues link the last (partial) helical
repeat to the PH domain. This is the most non-conserved region
of sequence between the β spectrins, and is a ligand binding
site. These residues in the long C-terminal form of βII spectrin
(residues 2087-2198) have a specific interaction with the
neuronal protein fodaxin, a protein which does not interact
with either the long or the short forms of βI spectrin (Hayes
and Baines, 1994; Hayes et al., 1997). The differential mRNA
splicing that gives rise to the long and short forms of βI spectrin
occurs about half way through the region (Winkelmann et al.,
1990a), so this region varies not just between products of
different spectrin genes, but also between different of products
of the same gene.
By contrast to the activities of long C-terminal forms, the
short C-terminal region of the erythrocyte βI spectrin has no
documented ligand binding activities; however multiple
residues unique to this short β chain are substrates for casein
kinase (Harris and Lux, 1980; Pedroni et al., 1993).
Phosphorylation of erythrocyte βI spectrin is associated with
control of erythrocyte mechanical function (Manno et al.,
1995). The long and short forms of βI spectrin therefore differ
in terms of ligand binding and regulatory properties.
In this paper, we consider whether βII spectrins, like βI, have
a short, functionally distinct, variant? Taking advantage of the
expressed sequence tag (EST) database, we have identified a
sequence that we show is contiguous with the βII gene. This
sequence encodes a variant that is structurally analogous to the
short C-terminal region of human erythrocyte βI. While this
work was in progress the sequences of two products of the
fourth β spectrin gene, ELFs 1 and 3, were described (Mishra
et al., 1998, 1999). The C-terminal regions of these proteins are
extremely similar in both nucleotide and amino acid sequence
to the ‘short’ variant of βII spectrin that we have identified. The
common C-terminal portions of products of βII and elf genes
seem to represent a novel structural domain characteristic of
products of these genes. This structural domain is rich in
potential phosphorylation sites, but is functionally distinct from
the ‘long’ form of βII spectrin in that it lacks the PH domain,
and also the site for interaction with the protein fodaxin (Hayes
et al., 1997). Antibodies selective for long and short forms of
βII spectrin reveal that while there is extensive overlap in the
intracellular location of the two proteins in muscle cells and
neurons, they are not always coincident. In particular skeletal
muscle and heart M line regions contain the short form; the
distal portions of cerebellar granule cell neurites are enriched
in the long form. Only the long form is retained in preparations
of post-synaptic densities. The respective C termini appear to
correlate with differential compartmentalization of isoforms.
We conclude that a recurring theme in the biology of most (if
not all) mammalian β spectrins is likely to be functional
variation mediated by variant C termini.
MATERIALS AND METHODS
Procedures and reagents
Standard molecular biology methods were used (Sambrook et al.,
1989) except where stated. Human skeletal muscle cDNA was
obtained from Cambridge Biosciences, UK. Human genomic DNA
was kindly given by Dr S. Eber (Georg-August-Universitat,
Gottingen, Germany). Unless otherwise indicated all chemicals and
reagents were obtained from Sigma (Poole, Dorset, UK). The antifodaxin monoclonal antibody, DR1, was used as described previously
(Hayes and Baines, 1994). FITC-conjugated AffiniPure donkey antiguinea pig IgG (Jackson Immunoresearch Laboratories, Inc) and
TRITC-conjugated goat anti-rabbit, TRITC-conjugated anti-mouse
IgG and FITC-conjugated anti-rabbit IgG (Vector Laboratories,
Peterborough, UK) were used as secondary antibodies for
immunofluorescence. Peroxidase-conjugated secondary antibodies for
immunoblotting were from Dako (Cambridge, UK).
Database searches and sequence alignments
Database searches were made using the NCBI BLAST server
available
over
the
World
Wide
Web
at
http://www.ncbi.nlm.nih.gov/BLAST/. Potential splice sites in
genomic sequences were identified using Splice Site Prediction by
Neural Network http://www.fruitfly.org/seq_tools/splice.html. GCG
Wisconsin Package version 8 (GeneticsComputerGroup, 1994) was
used for all other sequence analyses.
cDNA encoding the C-terminal region of the variant βII
spectrin
cDNA encoding the C-terminal region of the short variant of βII
spectrin was obtained using polymerase chain reaction (PCR). In each
PCR, one primer represented the known βΙΙ spectrin sequence, the
other was designed to be specific for the putative variant βII, based
on the EST sequences. Primer 1 represented the 5′ end of the known
C-terminal region. Primer 2 was designed from a consensus of EST
sequences (see Results) for the short variant, and is complementary
to sequence in the 3′ untranslated region. Primer 3 represented
sequence encoding the 14th triple helical repeat.
(a) PCR using primers 1 and 2
The sequence of the sense primer was as follows:
CGTAGGATCCGTGCGCAGACAGCAAG (primer 1). A BamHI
site (underlined) was included at the 5′ end to facilitate cloning.
The sequence of the antisense primer was GGAATTCCAGAGGATTTGGAAAGGG (primer 2), and included an EcoRI site
(underlined). These primers were used to amplify either human
skeletal muscle cDNA (2-10 ng per reaction, giving a 0.3 kb product)
or human genomic DNA (0.1 µg per reaction, giving a 1 kb product).
Amplification was performed using Taq polymerase (Promega) for
cDNA or Advantage cDNA polymerase (Clontech) for genomic DNA.
The PCR conditions were as follows: 1 cycle of 95°C for 5 minutes,
55°C for 1 minute and 72°C for 1 minute; 30 cycles of 95°C for 30
seconds, 55°C for 1 minute and 72°C for 1 minute; and 1 cycle of
95°C for 30 seconds, 55°C for 1 minute and 72°C for 5 minutes. The
products were ligated into the TA cloning vector pGEM-T Easy
βII spectrins 2025
(Promega) and transformed into E. coli JM 109. Representative
plasmids were isolated and sequenced on both strands in triplicate by
the Advanced Biotechnology Centre, Charing Cross and Westminster
Medical School, London.
(b) PCR using primers 3 and 2
The sequence of the sense primer (primer 3) was as follows:
TTAGGGATCCGCAGTCCAAAGTGGATAAAC, and included a
BamHI site (underlined). The antisense primer was the same as that
described above (primer 2). Advantage cDNA polymerase (Clontech)
was used according to the manufacturer’s recommendations and the
following three-step PCR protocol was carried out: 1 step of 94°C for
5 minutes; 5 cycles of 94°C for 30 seconds, 72°C for 6 minutes; and
25 cycles of 94°C for 30 seconds, 68°C for 6 minutes. The products
were ligated into the TA cloning vector pGEM-T Easy (Promega) and
transformed into E. coli JM 109. Representative plasmids were
sequenced on both strands by MWG-Biotech UK Ltd, Milton Keynes,
UK.
Preparation of short βII spectrin C-terminal fusion protein
To prepare a glutathione-S-transferase (GST) fusion protein of the
short βII spectrin C-terminal region, the 0.3 kb cDNA fragment was
ligated into the prokaryotic expression vector pGEX-2T (Pharmacia)
and transformed into E. coli JM 109. A 36 kDa GST-fusion protein
was induced and purified by glutathione affinity chromatography by
methods described for other GST-fusion proteins (Hayes et al., 1997).
The recombinant polypeptide was judged pure by the criterion of a
single band on 12% SDS-PAGE.
Recombinant fragments of long βI and βII spectrins
A GST-fusion protein of the long C-terminal region of βI spectrin was
prepared as previously described (Hayes et al., 1997). This protein
contains residues 2009-2329, and is designated GST-βIΣ2(2009-2329).
The fusion protein was cleaved by thrombin to liberate the 35kDa
βIΣ2(2009-2329) C-terminal polypeptide. GST was removed from the
cleavage mixture by glutathione affinity chromatography.
A GST fusion protein of residues 2087-2198 of the long form of
βII spectrin was prepared as previously described (Hayes et al., 1997).
This is designated GST-βIIΣ1(2087-2198). This contains the region of
βII spectrin that binds the axonal protein fodaxin.
A modified pGEMEX-1 plasmid containing cDNA encoding amino
acid residues 2060-2333 of the human long form of βΙΙ spectrin was
also used. Details of this plasmid and purification of the 30 kDa
recombinant protein, βΙΙ(2060-2333), from E. coli BL21 (DE3) pLysS
are given in Davis and Bennett (1994) and Hayes et al. (1997).
Northern blots
Total RNA was extracted from various tissues by acid guanidinium
thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi,
1987). Equivalent amounts of RNA (10 µg) were fractionated by
denaturing agarose gel electrophoresis, transferred to Hybond-N
(Amersham), fixed by ultraviolet illumination and processed for
northern blot analysis. cDNA was labelled by random priming using
hexaprimers and [α-32P]dCTP for 24 hours at 37°C. Labelled DNA
was heat denatured prior to using as a probe. Membranes were
prehybridised for 2 hours followed by overnight hybridisation with
the appropriate cDNA probe. Both steps were carried out at 65°C.
Subsequently, membranes were washed and the signal was visualized
using an Instant Imager (Canberra-Packard).
Production of antibodies
Antibody to the short form of βII spectrin was raised in guinea pigs
to a peptide antigen. The peptide NSRRTASDQPWSGL, which
represents a unique sequence at the very C-terminal of this spectrin
isotype, was synthesized. The peptide was conjugated to keyhole
limpet hemocyanin (KLH, Pierce, Chester, UK) via the crosslinker 1ethyl-3-[3-dimethylaminopropyl] carboiimide hydrochloride (EDC).
Peptide (2 mg dissolved in 0.1 M MES, pH 4.5) was incubated at room
temperature for 2 hours with 2 mg KLH and 0.5 mg EDC with
constant shaking. Non-conjugated peptide was separated from KLHconjugate using a 9.1 ml PD-10 Sephadex G25 desalting column
(Pharmacia) and filtered using a 0.2 µm Millipore filter. 200 µg of
peptide conjugate was mixed with Freund’s complete adjuvant for the
initial immunization and with Freund’s incomplete adjuvant for
subsequent booster injections at 3-week intervals. The serum from the
fourth boost was affinity purified on short βII spectrin C-terminal
fusion protein coupled to Activated CH Sepharose 4B (Pharmacia).
The affinity-purified antibody was diluted 1:200 for immunoblots and
1:50-1:100 for immunofluorescence.
Antibodies to the long form of βII spectrin were raised in rabbits
to recombinant βΙΙ(2060-2333) using an immunization protocol
essentially the same as described above. Antiserum was affinity
purified on βΙΙ(2060-2333) conjugated to Sepharose. It was used at 1:500
on immunoblots and for immunofluorescence at 1:50-1:100.
Electrophoresis and blotting methods
Various rat tissues were dissected and rapidly powdered in liquid
nitrogen in a mortar and pestle. Boiling 5× sample buffer (Laemmli,
1970) was added to the powdered tissue, centrifuged and the
supernatant used for SDS-PAGE (Laemmli, 1970) on 6% or 7%
polyacrylamide gels. Purified spectrin C-terminal proteins were
analysed by 12% SDS-PAGE. Immunoblot analysis was carried out
as described by Nicol et al. (1997).
Immunohistochemistry of muscle and cerebellar sections
0.5-1 µm semi-thin frozen sections of adult rat extensor digitorum
longus (EDL) or diaphragm were prepared and analysed as described
by Kordeli et al. (1998). Cerebellum or heart were processed
essentially as described previously (Rayner and Baines, 1989).
Cerebellar granule cells
Granule cells were cultured from 7-day-old rats by standard methods
(as given in Nath et al., 1996), and were used in immunofluorescence
after 14 days in culture. The cells were fixed in methanol at −20°C
for 20 minutes, and processed for immunofluorescence as above.
Preparation and use of affinity columns
Purified C-terminal GST-βIIΣ1(2087-2198) spectrin, C-terminal GSTβIIΣ2(2087-2167) spectrin (both obtained as described above), GST and
bovine serum albumin affinity columns were prepared using Activated
CH-Sepharose 4B (Pharmacia) according to the manufacturer’s
recommendations. These columns were kept at 4°C and used within
2 months of being made. Conditions for affinity chromatography were
the same as previously described (Hayes and Baines, 1994). Briefly,
an enriched preparation of rat brain fodaxin was dialysed into buffer
A (10 mM sodium phosphate, pH 7.5, 2 mM EGTA, 0.05% Tween
20, 0.5 mM DTT and 0.02% sodium azide), filtered using a 0.45 µm
pore filter and applied to the affinity column. The column was washed
with 30 column volumes of buffer A. Bound proteins were eluted in
buffer A plus 1.2 M KBr. The results were analysed by SDS-PAGE
followed by immunoblotting.
Preparation of post-synaptic densities
Post-synaptic densities were prepared from pig brain by the method
of Carlin et al. (1980).
RESULTS
Detection of an alternatively spliced form of βII
spectrin
The two splice variants of βI spectrin differ at a region towards
the C terminus after the last (partial) triple helical repeat, so
2026 N. V. L. Hayes and others
we reasoned that an analogous variation might occur in βII. To
purposes of this report we will continue to refer to variants of
test this hypothesis, the database of expressed sequence tags
βII spectrin with long and short C-terminal regions.
(dbEST) was examined.
The novel C-terminal region is strikingly similar to ELF1
dbEST was searched using tBLASTn (Altschul et al., 1990)
and ELF3, two products of elf, a different β spectrin gene
with residues 2087-2194 of human βII (βG) spectrin (GenBank
(Mishra et al., 1998, 1999). The nucleotide sequences were
M96803). These residues encode a
region that links between the helical (A) Segments 15-19 of long (βIIΣ1) and short (βIIΣ2) C-terminal variants.
repeating part of βII spectrin and the
1680 YAGLKDLAEERRGKLDERHRLFQLNREVDDLEQWIAEREVVAGSHELGQDYEHVTMLQER
PH domain. Five human and one rat βIIΣ1
βIIΣ2
1 YAGLKDLAEERRGKLDERHRLFQLNREVDDLEQWIAEREVVAGSHELGQDYEHVTMLQER
ESTs (GenBank entries zl37f06.s1,
zl37f06.r1, yi59b05.s1, vy96c01.r1, βIIΣ1
1740 FREFARDTGNIGQERVDTVNHLADELINSGHSDAATIAEWKDGLNEAWADLLELIDTRTQ
61 FREFARDTGNIGQERVDTVNHLADELINSGHSDAATIAEWKDGLNEAWADLLELIDTRTQ
vr25f01.r1, yp50d05.s1) indicated the βIIΣ2
existence of an alternatively spliced C
βIIΣ1
1800 ILAASYELHKFYHDAKEIFGRIQDKHKKLPEELGRDQNTVETLQRMHTTFEHDIQALGTQ
terminus. The ESTs represented clones βIIΣ2
121 ILAASYELHKFYHDAKEIFGRIQDKHKKLPEELGRDQNTVETLQRMHTTFEHDIQALGTQ
from a variety of tissues.
Primers for PCR were designed to βIIΣ1
1860 VRQLQEDAARLQAAYAGDKADDIQKRENEVLEAWKSLLDACESRRVRLVDTGDKFRFFSM
181 VRQLQEDAARLQAAYAGDKADDIQKRENEVLEAWKSLLDACESRRVRLVDTGDKFRFFSM
amplify part of the predicted coding βIIΣ2
region of this spectrin. One primer was βIIΣ1
1920 VRDLMLWMEDVIRQIEAQEKPRDVSSVELLMNNHQGIKAEIDARNDSFTTCIELGKSLLA
based on the known βII spectrin βIIΣ2
241 VRDLMLWMEDVIRQIEAQEKPRDVSSVELLMNNHQGIKAEIDARNDSFTTCIELGKSLLA
sequence (the 5′ end being codon 2087);
1980 RKHYASEEIKEKLLQLTEKRKEMIDKWEDRWEWLRLILEVHQFSRDASVAEAWLLGQEPY
the other was designed to hybridize with βIIΣ1
301 RKHYASEEIKEKLLQLTEKRKEMIDKWEDRWEWLRLILEVHQFSRDASVAEAWLLGQEPY
the EST-predicted sequence in the 3′ βIIΣ2
Spectrin repeats ←↓→ C-terminal region
untranslated region. Amplification of
βIIΣ1
2040 LSSREIGQSVDEVEKLIKRHEAFEKSAATWDERFSALERLTTLELLEVRRQQEEEERKRR
human skeletal muscle first strand βIIΣ2
361 LSSREIGQSVDEVEKLIKRHEAFEKSAATWDERFSALERLTTLELLEVRRQQEEEERKRR
cDNA gave a single product of the
2100 PPSPEPSTKVSEEAESQQQWDTSKGEQVSQNGLPAEQGSPRMAETVDTSEMVNGATEQRT
predicted size (310 bp) (GenBank βIIΣ1
421 PPSPEPSTKVSEEAESQQQWDTSKGEQVSQNGLPAEQGSPRVSYRSQTYQNYKNFNSRRT
AJ005694). Amplification of rat skeletal βIIΣ2
muscle, kidney and brain cDNA gave βIIΣ1
2160 SSKESSPIPSPTSDRKAKTALPAQSAATLPARTQETPSAQMEGFLNRKHEWEAHNKKASS
identical PCR products, except for a βIIΣ2
481 ASDQPWSGL*..................................................
single nucleotide difference in the
2220 RSWHNVYCVINNQEMGFYKDAKTAASGIPYHSEVPVSLKEAVCEVALDYKKKKHVFKLRL
3′ untranslated region (GenBank βIIΣ1
............................................................
AJ242018). The amino acid sequences βIIΣ2
of the novel βII C-terminal are identical βIIΣ1
2280 NDGNEYLFQAKDDEEMNTWIQAISSAISSDKHEVSASTQSTPASSRAQTLPTSVVTITSE
in rat and human and confirmed the EST βIIΣ2
............................................................
prediction.
2340 SSPGKREKDKEKDKEKRFSLFGKKK*
Amplification of cDNA, from the βIIΣ1
βIIΣ2
..........................
14th spectrin repeat (codon 1680) to the
3′ end of the new sequence, confirmed
that the cDNA derived from βΙΙ (βG)
spectrin; the nucleotide sequence was (B) Last helical repeat and C-terminal regions of βIΣ1, βIIΣ2, and ELF3.
identical to the human sequence
1 IKEKLLQLTEKRKEMIDKWEDRWEWLRLILEVHQFSRDASVAEAWLLGQEPYLSSREIGQ
between codons 1680 and 2140, and βIIΣ2
ELF3
1 IKEKLLQLTEKRKEMIDKWEDRWEWLRLILEVHQFSRDASVAEAWLLGQEPYLSSREIGQ
included the ankyrin binding site in the βIΣ1
1 IREKLQQVMSRRKEMNEKWEARWERLRMLLEVCQFSRDASVAEAWLIAQEPYLASGDFGH
15th repeat. After this the two sequences
61 SVDEVEKLIKRHEAFEKSAATWDERFSALERLTTLELLEVRRQQEEEERKRRPPSPEPST
show little identity, and the predicted βIIΣ2
61 SVDEVEKLIKRHEAFEKSAATWDERFSALERLTTLELLEVRRQQEEEERKRRPPSPDPNT
coding region of the new sequence stops ELF3
βIΣ1
61 TVDSVEKLIKRHEAFEKSTASWAERFAALEKPTTLELKE..RQIAE....RPAEETGPQE
after 28 further codons (Fig. 1A).
Because the novel C-terminal βIIΣ2
121 KVSEEAESQQQWDTSKGEQVSQNGLPAEQGSPRVSYRSQTYQNYKNFNSRRTASDQPWSG
121 KVSEEAES.QQWDTSKGDQVSQNGLPAEQGSPRVSYRSQTYQNYKNFNSRRTASDHSWSG
sequence represents the second subtype ELF3
115 EEGETAGEAPVSHHAATERTSPVSLWSRLSSSWESLQPEPSHPY................
of βII spectrin, it would be βIIΣ2 βIΣ1
spectrin in the nomenclature of βIIΣ2
181 L
Winkelmann and Forget (1993). Since ELF3
180 M
.
the long form of βII was the first to be βIΣ1
discovered, it is βIIΣ1. Because of the
relative order of discovery, this is Fig. 1. Sequence of the novel βII spectrin short C-terminal isoform, in comparison to (A) the
long C-terminal isoform of βII (βG) and (B) to the short C-terminal isoforms of βI spectrin and
opposite to the nomenclature of βI ELF3. In this figure, human βII and
βI sequences are marked as follows: short C-terminal form
spectrin where Σ1 is the short form. In of βII βIIΣ2; long C-terminal form of βII βIIΣ1; short C-terminal region of βI βIΣ1. Regions of
the nomenclature of Hu et al. (1992), the the sequence are indicated in the figure. Note that the novel C-terminal variant is identical to
short variant would be βG246. We feel the long C-terminal isoform of βII until residue 2140, after which they diverge. The novel Cthat the nomenclature for the non- terminal sequence is very similar (although not identical) to the C-terminal sequence of mouse
specialist is confusing, and for the ELF3, but dissimilar to the short C-terminal sequence of human βI spectrin.
βII spectrins 2027
Fig. 2. Genomic organization of exons encoding the short
C-terminal βII spectrin. To examine the genomic organization
of the two βII spectrin isoforms, PCR was used to amplify
human genomic DNA encoding the respective C termini. PCR
products were analysed by electrophoresis on 0.9% agarose
gels, which were stained with ethidium bromide. PCR
analysis of (A) DNA encoding the 3′ coding regions of the
long C-terminal isoform of βII (lane 1) or the short C-terminal
isoform of βII (lane 2) from human genomic DNA: 3 kb and 1
kb bands were obtained respectively. The 3 kb band was
isolated, and used as a template for PCR with primers for the
short C-terminal. A 1 kb band resulted (lane 3). These data
indicate that the 3′ coding region of the short C-terminal form
is contained within the region of the sbtb2 gene that contains
the exons encoding the 3′ coding sequence of the long C-terminal form. (B) Generation of long and short C-termini by differential exon usage.
Schematic diagram representing the genomic DNA sequence that encodes the two βII spectrin isotypes. An intron interrupts the coding region
between codons 2119 and 2120. An exon starting at 2120 reads through to the stop codon of the short C-terminal isoform (asterisk). A splice
donor site within this exon exists at codon 2140 (marked spl). Usage of this site removes the region containing this stop codon and joins to an
exon encoding the remainder of the C-terminal of the long isoform.
90% identical. Fig. 1B shows comparison of the amino acid
sequences of the novel C-terminal region with mouse ELF3
and human erythrocyte β spectrin (βIΣ1) C-terminal regions.
The novel βII C-terminal and the ELF3 C-terminal regions
seem to represent a common sequence domain for these two
spectrins, clearly distinct from the short βI spectrin C-terminal
region.
We also noted an EST from adult rat (GenBank AI030132)
that is 98% identical to the mouse elf3 3′ coding sequence, and
which almost certainly derives from the rat elf gene. This EST
is only 91% identical to the novel rat βII short sequence. This
is consistent with rats having both the sbtb2 and elf genes.
The novel βII C-terminal region is encoded by the
same gene as the long form of βII spectrin
The different C-terminal sequences of βI spectrin derive from
alternative mRNA splicing within an exon (Winkelmann et al.,
1990a). To determine if this was also the case with βII spectrin,
human genomic DNA was amplified by PCR using primer
pairs in which one primer encoded the common region of both
spectrins, and the other was specific for the 3′ coding regions
of either the long or short C-terminal regions (see Materials
and Methods). Amplification of genomic DNA encoding the
long form gave a product of approx. 3 kb (Fig. 2A, lane 1);
correspondingly, the primers representing the short form gave
a product of approx. 1 kb (Fig. 2A, lane 2). The 3 kb product
was purified and reamplified with primers for the short form.
This gave a product of approx. 1 kb (Fig. 2A, lane 3),
indistinguishable by electrophoresis and restriction mapping
from the product of direct amplification of the original genomic
DNA. The region encoding the short form is evidently
contained within the region of the sptb2 gene encoding the long
form.
The 1 kb product was sequenced (GenBank AJ238723;
shown schematically in Fig. 2B). The coding region of βII Cterminal region is interrupted between codons 2119/2120 by a
678 bp intron. The following exon encodes the C-terminal
region of the new form. Within this exon, a splice donor site
is predicted (TCCACGGGTTAGTTA – marked spl in Fig. 2B).
This is at the point in the sequence where the new and original
forms vary. We infer that, as with βI spectrin (Winkelmann et
al., 1990a), a splicing event can occur within the exon that
encodes the C-terminal region of the short form. The splicing
event removes the stop codon present in the new form, and
allows the translation of a longer form of βII spectrin.
Expression of the mRNA for the short form in rat
tissues
To investigate the expression of mRNA encoding the short Cterminal sequence in the tissues of rat, northern blot analysis
was used. Total RNA from a variety of tissues was hybridized
under stringent conditions with a 142 bp human cDNA probe,
which comprised a sequence unique to the short form and did
not overlap with the long form (codons 2144-2167 of the short
form). Fig. 3 shows that two transcripts of approx. 7.5 kb (a
strong signal) and 7.0 kb (a much weaker signal) were found
in multiple tissues, being especially prominent in cardiac and
brain tissue, but detectable in mesenteric arteries, aorta and
lung tissue. Note that the last three of these have a significant
smooth muscle component. For comparison, Hu et al. (1992)
detected widespread expression of an approx. 9.4 kb transcript
of the long form of βII (βG) spectrin. They found expression
in brain was relatively high while that in heart was much
lower: we have also confirmed this data using RT-PCR (not
shown).
Northern blotting also distinguishes expression of mRNA
encoding short C-terminal βII from that encoding ELF3. In
particular, elf3 mRNA (9.0 kb) is longer than the short βII, and
abundant in lung, kidney and liver (Mishra et al., 1999), in
which our northern analysis detected little or no short Cterminal βII mRNA.
The more sensitive RT-PCR method was also used to detect
expression of the short C-terminal variant. Low levels of
expression were detected in kidney, liver and intestinal
epithelia, possibly reflecting the smooth muscle component of
blood vessels in these tissues. Sequencing of RT-PCR products
confirmed that these were the short variant of βII spectrin and
not an elf gene product.
Antibodies to the two forms of βII spectrin
The sequences of the two forms of βII spectrins diverge at
amino acid residue 2139. The following 28 amino acids in the
short C-terminal βII spectrin variant are unique. To generate
an antibody against the short C-terminal form, a synthetic
2028 N. V. L. Hayes and others
Fig. 3. Northern blot analysis of short C-terminal βII spectrin
transcripts. Total RNA was prepared from the indicated rat tissues,
fractionated by denaturing agarose gel electrophoresis (10 µg RNA
per lane) and transferred to Hybond-N. The filter was probed with
32P-labelled cDNA encoding only the unique region of the short
isoform of βII spectrin. Bands of approx. 7.5 and 7.0 kb (indicated)
were detected in several tissues, the strongest expression being in
heart (all four quadrants) and brain.
peptide was prepared representing the C-terminal 14 residues
from this sequence (see Fig. 1 and Materials and Methods). A
guinea pig antiserum was prepared as indicated in Materials
and Methods, and affinity purified on a GST-fusion protein of
the C-terminal 80 residues. Specificity of this antibody for
the short form was confirmed by immunoblotting with
recombinant fragments of the long and short forms of βII, as
well as the long form of βI. GST-short βII C-terminal spectrin
fusion protein was specifically recognized by the anti-peptide
antibody (Fig. 4, middle panel, lane 1) but recombinant long
form βII fragment (GST-βIIΣ1(2087-2198)) was not (Fig. 4,
middle panel, lane 2); neither was any immunoreactivity
observed to recombinant βI long form fragment
(βIΣ2(2009-2329)) (Fig. 4, middle panel, lane 3).
Rabbit antiserum against the long C-terminal form of βII
spectrin was produced using recombinant polypeptide
representing the C-terminal region of the long form of βII
(βIIΣ1(2087-2198)). Antibody to the long form detects the
recombinant long form C-terminal fragment (recombinant
βIIΣ1(2087-2198)) (Fig. 4, right hand panel, lane 2) but not the
short C-terminal fragment (lane 1), even though the fusion
proteins share a sequence of 42 amino acids at their respective
N termini (see Fig. 1). The antibody to the long form also
showed no reactivity with the C-terminal fragment of the long
form of βI spectrin (recombinant βIΣ2(2009-2329)) (lane 3).
The antibodies therefore recognize unique sequences in these
spectrin isotypes and can therefore distinguish between the two
alternatively spliced forms of βII spectrin. No reaction was
observed either by western blotting or immunofluorescence
with erythrocyte spectrin (βIΣ1 – data not shown).
Bearing in mind the extent of sequence identity between the
Fig. 4. Immunoblot analysis of antibodies to βII spectrin isotypes. The
figure shows replicate samples of recombinant β spectrin C-terminal
fragments after electrophoresis on 12% SDS-polyacrylamide gels.
Gels were either stained with Coomassie Blue (left), or transferred to
nitrocellulose paper and probed with antibodies to the short C-terminal
form (middle) or to the long C-terminal form of βII (right). The protein
samples were (1) GST-βIIΣ2(2087-2167) representing the C terminus of
the short isoform of βII (2) GST-βIIΣ1(2087-2198) representing the C
terminus of the long isoform of βII and (3) βIΣ2(2009-2329) representing
the C terminus of the long isoform of βI. The two anti-βII spectrin
antibodies appear specific for their respective isotypes.
short C-terminal βII spectrin variant and ELF1/3, it seemed
likely that antibodies to the short C-terminal βII spectrin
variant might be cross-reactive between these antigens. A
synthetic peptide was prepared using the equivalent sequence
of ELF1/3. This displaced approx. 60% of the reactivity from
GST-βIIΣ2 fusion protein in ELISA (not shown). This
indicates that antibodies to the short form will show some
cross-reactivity with ELF1/3. However, when we attempted to
detect the 27 kDa ELF1 protein that has been reported in brain,
kidney and liver using immunoblots (not shown) no reactivity
was found, indicating that for practical purposes this antibody
does not efficiently recognize the ELF proteins.
Using immunoblots calibrated with the GST-short βII Cterminal construct, the abundance of the short form of βII
spectrin was measured at approximately 2.5 pmol/mg protein
in a brain membrane fraction. This compares with estimates of
total spectrin content in brain membrane fraction of 30 pmol
spectrin tetramer/mg protein (Davis and Bennett, 1984).
Comparison between the distribution of the two Cterminal variants in rat muscle and nervous tissues
The distributions of the short and long C-terminal variants were
investigated in rat tissues (Fig. 5) by immunoblot.
Immunodetection using antibodies to the long form revealed
a single approx. 280 kDa band in heart, hind limb skeletal
muscle, forebrain, cerebellum and spinal cord (Fig. 5, middle
panel, lanes 1-5). Antibodies against the short C-terminal
variant detected a strong band at approximately 240 kDa in
heart (Fig. 5, lane 1, right hand panel). In hind limb skeletal
muscle (lane 2) a single 240 kDa immunoreactive product was
observed, but in forebrain (lane 3) only a single 270 kDa band
was observed. In the cerebellum (lane 4) two bands of 240 kDa
and 270 kDa were seen; in spinal cord (lane 5) only the 240
kDa band was observed. All immunoreactivity disappeared if
100-fold excess of the peptide antigen was present in the
βII spectrins 2029
Fig. 5. Immunoblot analysis of βII spectrin isotypes in rat
muscle and neuronal tissues. Replicate 6% SDSpolyacrylamide gels of tissue homogenates (lane 1, heart;
lane 2, skeletal muscle; lane 3, forebrain; lane 4,
cerebellum; lane 5, spinal cord) were eitherstained with
Coomassie Blue (left) or transferred to nitrocellulose paper
and probed with antibodies to the long C-terminal form
(middle) or to the short C-terminal form of βII (right).
Antibodies to the long C-terminal region of βII detect a 270
kDa antigen in each of the tissues. Antibodies to the short
C-terminal region of βII detect an approx. 240 kDa band in
heart, skeletal muscle cerebellum and spinal cord; an
additional band at approx. 270 kDa is detected in forebrain
and cerebellum.
primary antibody (data not shown). These data indicate that
both variants of βII spectrin can be present in the same tissue.
Selective incorporation of the short C-terminal βII
spectrin variant into muscle M line regions, but both
forms are Z line components
The immunoblotting data suggested that both variants are
expressed in heart, skeletal muscle and nervous tissue. Indirect
immunofluorescence was used to compare the distribution of
the two βΙΙ spectrins in rat skeletal muscle (Fig. 6) and in
ventricular heart muscle (Fig. 7).
In longitudinal sections of skeletal muscle (extensor
digitorum longus; Fig. 6A-F), both βII spectrin variants were
at the Z-line level, as indicated by counterstaining with
antibodies to α-actinin. In addition punctate staining with
antibodies to the long form was also observed. As with heart,
antibodies to the short form showed clear labelling throughout
the depth of the M-line level; no M-line labelling was detected
for the long form (Fig. 6C,F, insets). Specificity of the
antibodies on these sections was confirmed by preabsorption
of the antibodies with relevant antigen, which abolished
specific staining (not shown). In cross-sections of diaphragm
(not shown) neither βII spectrin was especially concentrated at
or near the plasma membrane, and in particular, no labelling
was detected at neuromuscular junctions (NMJs), again in clear
distinction to βI spectrin.
Both βII spectrin antigens were detected at the Z line in
cardiac tissue (Fig. 7). In confocal microscopy, their
distribution overlapped with anti-α-actinin (a wellcharacterised Z disc component). However, in addition to Zline staining, a longitudinal myoplasmic distribution was
observed for both βII polypeptides, although this was more
obvious with antibodies to the long form. The most striking
difference was selective staining of the central region of the
sarcomere with antibodies to the short form: this staining is
coincident with the M line.
It was unlikely in heart that either form was associated with
the T-tubule system, as a similar regular striated staining
pattern was observed in cardiac atrial tissue, which does not
contain many T-tubules (data not shown). No sarcolemmal or
costameric labelling was detected with either antibody (not
shown), clearly distinguishing βII from βI spectrin in muscle
(Wood and Slater, 1998; Zhou et al., 1998).
In some sections immunoreactivity with blood vessels was
detected. In these cases, antibodies to both the βII isotypes
reacted with the smooth muscle cells lining the vessels, but not
with erythrocytes within the blood vessels.
Retention of the short C-terminal variant near the
cell soma of cerebellar granule neurons
In frozen sections of cerebellar molecular and granule cell layers
(Fig. 8A), the long C-terminal variant was detected in the granule
cell layer, the Purkinje cells and the parallel fibres of the
molecular layer. By contrast, little if any immunoreactivity for
the short βII C-terminal variant was found in the parallel fibres,
although cerebellar granule cells and Purkinje cell bodies were
recognized by the antibody (Fig. 8B). Purkinje cell dendrites
were not stained. This indicates that the short βII C-terminal
variant is probably restricted to regions close to cell bodies. In
the white matter tracts, axons were abundantly labelled with
antibodies to the long form, but staining with antibodies to the
short form was comparatively weak (not shown).
In cultures of primary rat cerebellar granule neurons (Fig.
8C-E) both isoforms were present in individual cells, but the
short C-terminal βII variant had a more restricted distribution
than the long form. The long C-terminal form was present in
many neurites, but the short βII variant was predominantly in
the cell soma, with a distribution in neurites primarily limited
to their proximal segments. Many fine processes were noted in
the culture that only reacted with antibodies to the long form,
and not at all with the short βII variant. The distribution of the
short βII variant in cell bodies appeared very diffuse, and was
not particularly associated with plasma membranes. In both
sections of cerebellum and primary granule neuronal cultures,
the short C-terminal variant was restricted more closely to the
cell body than the long C-terminal variant.
Fodaxin does not interact with the C-terminal region
of the short form of βII spectrin
Fodaxin is an axonal protein that interacts with the long Cterminal form of βII spectrin (Hayes et al., 1997), and is also
present in cerebellar parallel fibres and white matter axons
(Rayner and Baines, 1989). Fodaxin therefore codistributes
with the long, but not the short C-terminal βII. The region
of the long form of βII spectrin that interacts with fodaxin
comprises residues 2087-2198, i.e. the region of variation
2030 N. V. L. Hayes and others
between the long and short C-terminal forms. Given this, it
was important to test whether or not the short form retained
the fodaxin binding activity. To assay binding, affinity
chromatography was used with affinity columns made from
recombinant C-terminal fragments of each isoform. An
enriched preparation of fodaxin from rat cerebral cortex
(Hayes et al., 1997) was prepared for affinity
chromatography and applied to an affinity column made by
coupling a GST fusion protein of the short C-terminal region
to Sepharose (Fig. 9). Fodaxin was detected using the
monospecific monoclonal antibody DR1. Fig. 9A shows that,
as expected, fodaxin interacted with the affinity column
made from the long C-terminal form. Fodaxin did not appear
in the breakthrough fractions from this column, and was only
recovered by eluting the column with a dissociating buffer
containing 1.2 M KBr. By contrast, when fodaxin was
applied to the affinity column of the short C-terminal form
(Fig. 9B), it was found in the breakthrough fractions only
and was not detectable in the wash or fractions that had been
eluted with 1.2 M KBr. These results indicate that the Cterminal region of the short form does not interact
with fodaxin under these conditions and
demonstrate a difference in activity between the
C-terminal regions of the two βII spectrin
variants.
The short βII variant is not retained in postsynaptic density preparations
One further comparison of the properties of the
two βII spectrin variants was made by examining
their relative retention in preparations of
postsynaptic densities (PSDs). PSDs are enriched
in spectrin (Carlin et al., 1983), including βII
spectrin, which appears to form part of an
apparatus bridging certain receptors to
cytoplasmic actin (Wechsler and Teichberg,
1998). PSD preparations from pig brain were
compared for their content of the long and short
forms of βII spectrin (Fig. 10). Coomassie Blue
stained gels of equal protein loadings of brain
homogenate and a PSD preparation are shown
in Fig. 10, lanes 1 and 2. Corresponding
immunoblots of PSDs were probed with
antibodies to the two forms (long form, lanes 3
and 4; short form, lanes 5 and 6), with the known
PSD protein PSD-95 as a standard PSD marker
(lanes 7 and 8). Note the enrichment of PSD-95 in
the PSD preparation. The 280 kDa band of the
long C-terminal βII variant was readily detected
in the PSD preparation, but the short C-terminal
variant was clearly depleted.
The long C-terminal region of βII spectrin
correlates with targeting to and/or retention in
PSDs, and these data suggest that there are PSDspecific interactive ligands for this variant.
DISCUSSION
In this paper we describe a novel variant of βII
spectrin. This variant is derived from the same gene
(sptb2) as the previously published βII (βG) spectrin (Hu et al.,
1992; Ma et al., 1993), but differs through alternative mRNA
splicing near the 3′ end of the coding region. The predicted
protein sequence shown in Fig. 1 is shorter than the first βII
sequence to be published, and contains no PH domain. The site
of variation occurs about half way between the last (partial)
triple helical repeat and the PH domain. The new sequence is
only 28 amino acid residues long, and is generally hydrophilic
in character. It is comparatively rich in amino acid residues
carrying hydroxyl groups (ser, thr and tyr: 10 of these in total).
Strikingly, the C-terminal region from the end of the helical
repeats to the short C terminus is nearly identical in amino acid
sequence to the corresponding regions in two products of the elf
gene, the polypeptides ELF1 and ELF3 (Mishra et al., 1998,
1999) (Fig. 1). Indeed the nucleotide sequences are about 90%
identical. There is no question, though, that the cDNA that we
have characterized comes from the gene that encodes βII
spectrin (the sptb2 gene). First, the sequence of the region of the
coding sequence upstream of the variant region is absolutely
identical to the published βII sequence, but only about 90%
Fig. 6. Immunofluorescence analysis of βII spectrin isoforms in rat skeletal
muscle. 0.5 µm semi-thin frozen longitudinal sections of EDL skeletal muscle
were double-labelled with antibodies to (A) the short C-terminal variant of βII
spectrin or (D) the long C-terminal variant of βII spectrin, and compared with
antibodies to (B,E) α-actinin. The insets in each image show higher magnification
views of the regions outlined in the main image. There is extensive coincidence of
the two stains for both isoforms of βII spectrin and α-actinin in the Z-line region
(yellow in merged image, C,F) but there is selective staining for the short Cterminal form at the level of the M line (arrows) and in longitudinal elements (C).
Νo staining for the long C-terminal variant is detectable at the level of the M-line.
Scale bar, 10 µm.
βII spectrins 2031
Fig. 7. Immunofluorescence analysis of βII spectrin isotypes in rat
cardiac muscle. Longitudinal frozen sections of formaldehyde-fixed
cardiac muscle (4 µm thickness) were labelled with antibodies to (A)
the short C-terminal variant of βII spectrin or (B) the long C-terminal
variant of βII spectrin. As with skeletal muscle (Fig. 6), the major
striations labelled with both antibodies are Z lines. In addition,
labelling at the M-line level was noted selectively with antibodies to
the short form, indicated by arrowheads in A. Scale bar, 10 µm.
identical to elf. Second, the coding region for the novel C
terminus is within a 3 kb genomic DNA region that contains
sequence encoding both C termini (Fig. 2). The exon that
encodes the end of the novel reading frame contains within it a
predicted splice site at the point of sequence variation between
the long and short forms. This is similar to the exon organization
in the gene encoding βI (Winkelmann et al., 1990b). Third, a rat
EST that appears to encode an elf product can be distinguished
from the rat cDNA sequence for our variant βII (noted in
Results). In comparison with βII variation, no elf gene product
has yet been published that has a PH domain.
A major concern in our investigations of the short βII Cterminal mRNA and polypeptide is the degree of similarity to
elf gene products. It was conceivable that our investigations of
these might have been complicated by cross-hybridization of
cDNA probes, or cross-reaction of antibodies. As indicated in
Results, we view our probes as being specific for sptb2
products. Our RT-PCR is clearly specific for cDNA encoding
βII since we have sequenced representative PCR products. In
northern blots (Fig. 3), the size of the mRNA encoding the βII
spectrin short C-terminal variant was smaller than the reported
size (Mishra et al., 1999) of ELF3 mRNA, and the tissue
distribution clearly differed. In particular, where heart contains
little ELF3 mRNA, this is one of the most abundant sources of
the short βII C-terminal variant. Antibodies to the short βII
variant show limited cross-reactivity with an ELF peptide in
ELISA, but do not detect ELF1 in blots of tissues, nor do they
crossreact detectably with βI or the long C-terminal form of
βII (Fig. 4). All these data indicate that we have only used
probes specific for the relevant spectrin.
The reaction of two bands in brain tissue with the antibodies
to the short C-terminal variant was noted in Fig. 5: one was
approx. 240 kDa, the other approx. 270 kDa. The 240 kDa form
was also found in other tissues, and its size correlates well with
the predicted size of the short-terminal variant βII polypeptide
Fig. 8. βII spectrin isotypes in
cerebellum and cultured
cerebellar granule cells. The
distributions of (A) long and
(B) short C-terminal βII
isoforms in rat cerebellum
granule and molecular layers
were analysed by
immunofluorescence. Note that
both isoforms are present in
cell bodies in the granule cell
layer (gc) and in Purkinje cell
bodies (pc). However, the shorter isoform is not detectable in the
parallel fibres (pf) of the molecular layer, unlike the long form.
Primary cultures of rat cerebellar granule cells were analysed by
immunofluorescence using (C) antibodies to the long C-terminal
region of βII or (D) antibodies to the short C-terminal region of βII
followed by FITC-conjugated or TRITC-conjugated secondary
antibodies, respectively. A merged image of C and D is shown in (E).
Note that the long C-terminal isoform is much more extensively
distributed in the neuronal processes than the short C-terminal form
(C compared to D) and that the short form is more prominent in the
perikarya and neurite proximal region than in the neurite distal
region.
(246 kDa). The molecular identity of the approx. 270 kDa band
is not clear, but it is not likely to be ELF3, for the reasons noted
above. One possibility is that it represents another splice
variant of βII spectrin that has not yet been characterized.
Immunofluorescence (Figs 6-8) indicates that the two βII
spectrin isoforms have a distribution that overlaps in any one
cell, but is not identical. In skeletal and cardiac muscle, both
isoforms are present at the level of the Z line, but only the Mline level contains the short C-terminal variant (Figs 6, 7). Cterminal variation therefore correlates with intracellular
targeting.
2032 N. V. L. Hayes and others
Fodaxin eluted (arbitrary units)
Fig. 9. The long, but not the short, C(A) βIIΣ 1 segment 19L
(B) βIIΣ 2 segment 19
terminal isoform of βII spectrin interacts
with fodaxin. The interaction of fodaxin and
100
100
the two βII spectrins was assayed by affinity
chromatography. Affinity columns made
80
80
from the indicated spectrin constructs were
used to test the interaction with sheep brain
60
60
fodaxin. 0.5 ml of fodaxin was loaded onto
(A) a column of a recombinant fragment of
40
40
the long C-terminal region of βII spectrin
coupled to Sepharose or (B) a column of a
20
20
recombinant fragment of the short Cterminal region of βII spectrin coupled to
0
0
Sepharose. The start of the x axis (volume)
0
10
20
30
40
0
10
20
30
40
on these graphs marks the point of loading.
The sample was allowed to run into the
Volume (ml)
Volume (ml)
column, which was washed with binding
buffer until the point indicated by the arrow. At this point the buffer was changed to a dissociating buffer containing 1.2 M KBr. Fractions
eluting from the column were monitored by immunoblotting and densitometry. Fodaxin is retained only on the long C-terminal region affinity
column, not on the short C-terminal region affinity column, or on other control columns (not shown), indicating selective binding of fodaxin to
one spectrin isoform.
The distribution of the βII spectrin isoforms in muscle
clearly differs from the βI isoform. The βI spectrin is
concentrated at the sarcolemma (e.g. Porter et al., 1997; Zhou
et al., 1998) and enriched in neuromuscular junctions (Wood
and Slater, 1998). The novel βII spectrin we describe here is
enriched in internal structures, not sarcolemma or
neuromuscular junctions (NMJs). We specifically examined
NMJs in diaphragm using α-bungarotoxin and were unable to
detect colocalizing βII short C-terminal variant. No
immunofluorescence from erythrocytes in blood vessels in our
sections was detected with this antibody, indicating no crossreactivity with erythrocyte βI spectrin. Zhou et al. (1998)
reported little reaction of anti-β non-erythroid spectrin with
internal structures of muscle; however, a number of other
authors (e.g. Isayama et al., 1993) have reported spectrins
associated with internal muscle structures. Since the exact
isotype specificity of the antibodies used by Zhou et al. (1998)
was not reported in their paper, it is difficult to make
comparisons between their results and those reported here.
Nervous tissue is an abundant source of both isotypes of βII
spectrin, and tissue with a muscle component (especially skeletal
muscle and heart) contains readily detected short C-terminal βII
variant (Figs 3, 5). Although the short variant is detectable by
RT-PCR in tissues characterized by the presence of, for example,
polarized epithelia (kidney or intestinal epithelium) it is a minor
product in these tissues. The short βII is expressed most
abundantly in electrically excitable tissue. The short C-terminal
βII variant exists at about 2.5 pmol/mg protein in brain
membrane fraction (see Results). Total brain spectrin tetramer is
estimated at 30 pmol/mg, i.e. total β spectrin is about 60
pmol/mg. The short βII is therefore about 4% of total β spectrin.
Bearing in mind the restricted nature of its distribution, and that
there are four β spectrin genes, the fact that it is a comparatively
small proportion of total β spectrin is not surprising. We might
estimate that in the cerebellar granule/Purkinje cell layer, the
short variant is perhaps three to four times more concentrated
than in total homogenate, i.e. representing 12-15% of total β
spectrin, a significant proportion in the context of a cell layer
documented to have products of βI and βIII gene products, as
well as another variant of βII.
Fig. 10. Enrichment of the long C-terminal form of βII spectrin in
post-synaptic density preparations, but depletion of the short form.
The presence of the two βII spectrin isoforms in preparations of postsynaptic densities was investigated by immunoblot. The figure shows
replicate gels of total pig brain homogenate (1, 3, 5, 7) and postsynaptic densities (2, 4, 6, 8). Total protein loadings in each lane
were the same. The gels were stained with Coomassie Blue (lanes 1
and 2), or transferred to nitrocellulose and probed with antibodies to
the long form of βII spectrin (lanes 3, 4), antibodies to the short form
of βII spectrin (lanes 5, 6) or to the known post-synaptic protein
PSD-95 (lanes 7, 8). Note the enrichment of PSD-95 in the
preparation. The long isoform of βII spectrin is moderately enriched
in the PSD preparation, relative to starting homogenate, but the short
isoform is strongly depleted. The positions of marker proteins (kDa)
are shown.
In cerebellum, the distributions of both the long and short
βII isoforms differ from each other and the long βI spectrin.
Malchio-Dialbedi et al. (1993) reported that the long Cterminal isoform of βI, βIΣ2 spectrin, was enriched in granule
cells and Purkinje cell dendrites; it was abundant in PSDs. In
contrast and in agreement with the overall distribution of βII
βII spectrins 2033
reported by Clark et al. (1994) the long βII was abundant in
axons (cerebellar parallel fibres, cultured granule cell neurites).
The short βII had a more restricted distribution: the cell bodies
of proximal neurite segments of granule cells, and Purkinje cell
bodies were positive for the short βII; PSDs were depleted of
short βII. In this context, it is of interest to note that PSDs
contain N-methyl-D-aspartate (NMDA) receptors. NMDA
receptors have been reported to bind βII spectrin (Wechsler and
Teichberg, 1998). Since the long, but not the short, βII isoform
is present in PSDs, it may be that NMDA receptors form one
of the classes of direct membrane binding site for the Cterminal region of βII spectrin reported by Lombardo et al.
(1994) and Davis et al. (1994). βIII spectrin has been reported
to be associated with presynaptic structures (Sakaguchi et al.,
1998). Different β spectrin complements seem to characterize
the various compartments of cerebellar neurons.
The intra-neuronal compartmentation of the two βII forms
correlates also with their comparative binding activities.
Fodaxin is an axonal protein of the membrane skeleton in many
neurons (Rayner and Baines, 1989). In particular it is present
in the parallel fibers of the cerebellar molecular layer that are
axons of granule cells (Hayes and Baines, 1994; Rayner and
Baines, 1989). Fig. 9 indicates that only the long form can bind
fodaxin; this correlates with the enrichment of this form in
parallel fibres. Correspondingly, the short βII is restricted
primarily to cell body structures, and does not bind fodaxin.
βIΣ2 also does not bind fodaxin (Hayes et al., 1997), again
corresponding with the lack of colocalisation. All these data
indicate an exquisite level of compartmentation of spectrin
isoforms in the nervous system, which correlates with the
selective nature of their interactions.
All these observations point to the variant C-terminal regions
correlating with differential subcellular targeting. The simplest
deduction is that the C termini act as targeting modules for their
respective polypeptides. This has a precedent in the
observation that a β spectrin associated with Golgi is targeted
to it by interactions of the PH domain (Godi et al., 1998). It
will be important to test the hypothesis that the short βII
spectrin C-terminal functions as, for example, an M-line
targeting module in striated muscle. Likewise, it will be
interesting to test if the βII long C-terminal is a PSD-targeting
module.
This work was supported by a project grant to A.J.B. from the
Biotechnology and Biological Sciences Research Council, and
equipment grants from the Wellcome Trust. C.S. is the recipient of a
BBSRC Special Studentship. Work in V.O.’s laboratory is supported
by the British Heart Foundation. A.J.B. and E.K. acknowledge a
British Council Alliance grant. This work benefited from the
BBSRC’s Seqnet facility. We thank Ms Shweta Singh for supplying
cerebellar granule cell cultures.
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