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Molecular and Cellular Biology, November 2000, p. 8209-8219, Vol. 20, No. 21
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Profilin II Is Alternatively Spliced, Resulting in Profilin
Isoforms That Are Differentially Expressed and Have Distinct
Biochemical Properties
Anja
Lambrechts,1
Attila
Braun,2
Veronique
Jonckheere,1
Attila
Aszodi,2
Lorene M.
Lanier,3
Johan
Robbens,1
Inge
Van
Colen,1
Joël
Vandekerckhove,1
Reinhard
Fässler,2 and
Christophe
Ampe1,*
Department of Biochemistry, Ghent University
and Flanders Interuniversity Institute for Biotechnology, 9000 Ghent,
Belgium1; Department of Experimental
Pathology, Lund University, 22 1 85 Lund,
Sweden2; and Department of Biology,
Massachusetts Institute of Technology, Cambridge, Massachusetts
02139-43073
Received 9 March 2000/Returned for modification 24 May
2000/Accepted 8 August 2000
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ABSTRACT |
We deduced the structure of the mouse profilin II gene. It contains
five exons that can generate four different transcripts by alternative
splicing. Two transcripts encode different profilin II isoforms
(designated IIa and IIb) that have similar affinities for actin but
different affinities for polyphosphoinositides and proline-rich
sequences. Profilins IIa and IIb are also present in humans, suggesting
that all mammals have three profilin isoforms. Profilin I is the major
form in all tissues, except in the brain, where profilin IIa is most
abundant. Profilin IIb appears to be a minor form, and its expression
is restricted to a limited number of tissues, indicating that the
alternative splicing is tightly regulated. Western blotting and
whole-mount in situ hybridization show that, in contrast to the
expression of profilin I, the expression level of profilin IIa is
developmentally regulated. In situ hybridization of adult brain
sections reveals overlapping expression patterns of profilins I and IIa.
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INTRODUCTION |
Changes in both cell shape and
motility in response to extracellular signals require mechanical forces
from the actin cytoskeleton. The formations of lamellipodia, focal
contacts, the contractile ring during cytokinesis, and neuronal growth
cone motility depend on the controlled
polymerization-depolymerization of actin filaments (7,
40). The regulation of these processes is accomplished by many
actin binding proteins that act at different points in time and space,
depending on the extracellular signals (44).
Profilin is a small, ubiquitous actin binding protein, thought to be a
key regulatory of actin dynamics in living cells (8, 41). In
vitro experiments have shown that profilin acts as an actin
monomer-sequestering protein when barbed ends of filaments are capped
(e.g., by gelsolin). When the capping protein is removed, polymerization of actin can occur, and actin-profilin complexes can add
to the fast-growing ends, thereby enhancing actin polymerization in the
presence of thymosin 
4 (22, 34).
Profilin can bind other molecules including phosphatidylinositol
4,5-bisphosphate (PIP2) (28) and proline-rich
domains of several proteins like the vasodilator-stimulated
phosphoprotein (VASP), Enabled (Ena), mammalian Ena (Mena),
aczonin, the Wiskott-Aldrich syndrome protein (WASP), its neural
homologue N-WASP, and mammalian Diaphanous (p140mDia) (1, 2, 13,
38, 45, 46, 48). In lower eukaryotes, many other proline-rich
proteins have been identified as profilin ligands, such as
formin-homology proteins (47). The actin-related
protein-2/3 complex (Arp2/3 complex), composed of seven different
polypeptides, was originally identified as a profilin binding complex
(29).
Until 1993, only one profilin isoform was known to be present in
mammalian cells. Honoré and coworkers (20) discovered a second profilin gene in a random cDNA cloning project. The open reading frame predicted a protein with a high similarity to profilin I
(62.1% identity), and therefore the protein was designated profilin II. Northern blot analysis showed that the expression levels of the two
profilins were complementary in many tissues. Profilin I expression was
highest in placenta, lung, liver, and kidney, while profilin II
transcripts were most abundant in brain, skeletal muscle, and kidney.
However, three transcripts of different lengths that hybridized with
the coding as well as noncoding regions of the profilin II cDNA were
observed for profilin II.
In 1995, two papers reported conflicting data on the biochemical
characterization of profilin II. We purified and characterized profilin
II from bovine brain, taking advantage of the low expression of
profilin I and high expression of profilin II in this organ (25). In an independent study, Gieselmann and coworkers
analyzed the human recombinant profilin II form, produced in
Escherichia coli (14). We found that bovine
profilins I and II have similar affinities for actin and that profilin
I has a higher affinity for PIP2 while profilin II binds
more strongly to poly-L-proline. We subsequently showed
that profilin II recruits VASP from bovine brain extracts, while
profilin I does not. In contrast, profilin I prefers binding to
PIP2 over poly-L-proline in a competition assay
(26). The results of Gieselmann and coworkers
(14) were strikingly different and showed that human
profilins I and II have similar affinities for PIP2 and
poly-L-proline, though profilin I has a five-times-higher
affinity for actin.
The data that we present in this paper show that the two groups worked
with different profilin II isoforms (designated profilin IIa and
profilin IIb) that are generated by alternative splicing. The different
carboxy-terminal parts confer different biochemical properties on the
proteins. The genomic sequence of profilin II reveals a complex gene
structure. In addition to the four exons necessary for the formation of
the two profilin isoforms, a fifth (noncoding) exon is present,
potentially giving rise to truncated profilin forms. The profilin IIb
message is detected in only a few tissues and is present at much lower
levels than is profilin IIa. Furthermore, we provide evidence that the
expression patterns of profilin I and of profilin IIa are different
during development and that profilin IIa is the major form in neural
tissues. In the adult brain, expression of profilin I and that of
profilin II are overlapping.
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MATERIALS AND METHODS |
Recombinant DNA methods.
Total RNAs from various adult mouse
and rat tissues and from mouse developmental stages were prepared by
the guanidinium isothiocyanate method (11). Oligo(dT)-primed
reverse transcriptions (RT) were carried out using the Superscript
Preamplification system (mouse) or Thermoscript R (rat) (both from
GIBCO BRL). PCRs were performed with Taq DNA polymerase
(GIBCO BRL) using the primers listed in Table
1. The amplified products were separated
on 2% agarose gels and stained with ethidium bromide. The intensities
of the bands were quantified with the Gel Pro Analyzer (Techtum)
package. The same amount of cDNA and the same PCR protocol were used to amplify -actin as a control. 5' rapid amplification of cDNA ends (RACE) of the profilin II cDNA was done using mouse brain Marathon RACE-ready cDNA (Clontech) and the Advantage Klen Taq Polymerase mix
(Clontech) according to the manufacturer's instructions. The first
amplification was carried out with the adapter primer AP1 (Clontech)
and the gene-specific primer exon 3 reverse (Table 1). The second,
nested amplification was performed using primer AP2 (Clontech) and the
gene-specific primer exon 2 reverse.
Nucleotide sequencing of genomic DNA and mouse PCR products was
performed with the Thermo Sequenase Dye Terminator Cycle Sequencing
premix kit (Amersham). Sequence reaction products were resolved
on an
ABI Prism 377 Automated Sequencer (Applied Biosystems).
Nucleotide
sequence analysis was performed using the GCG software
package
(Genetics Computer Group). Dideoxy DNA sequencing on human
and rat PCR
products was done using the T7 sequencing kit from
Pharmacia on Wizard
(Promega)-purified plasmid DNA with M13 forward
and reverse
primers.
To clone the profilin II gene, we screened a P1-derived artificial
chromosome library containing mouse genomic DNA (United
Kingdom HGMP
Resource Centre, Cambridge, United Kingdom) using
a
32P-labeled
NcoI-
PstI fragment of a
profilin II mouse expressed
sequence tag (EST) clone (
AA032658,
obtained from the IMAGE
consortium). One positive clone was identified
and digested with
EcoRI and
BamHI. Fragments
hybridizing with the profilin II cDNA
were subcloned into pBluescript
KS(+).
Biochemical methods.
We cloned the profilin cDNAs in the
NcoI/BamHI sites of the pEt11d vector. This does
not result in additional amino acids in the expressed proteins. The
proteins were expressed in E. coli strain MC1061 harboring
pT7POL26 (32). When the cells reached an optical density at
600 nm of 1, we added IPTG
(isopropyl--D-thiogalactopyranoside) to a final
concentration of 1 mM, and the cells were induced for 3 to 4 h.
Cell pellets were resuspended in 20 mM Tris-HCl (pH 8.1)-1 mM EDTA-5
mM dithiothreitol (buffer A) supplemented with protease inhibitors.
Subsequently, we lysed the cells with a French press and cleared the
lysate by ultracentrifugation for 1 h at 100,000 × g.
We loaded the cleared lysate on a
poly(L-proline)-Sepharose column, and after washing the
column with buffer A, we eluted the proteins with increasing
concentrations of urea (3, 5, and 8 M in buffer A). Bovine profilin IIa
was purified from bovine brain (25). 
-Actin was purified
from rabbit skeletal muscle as described previously (35,
43). Cys-375-pyrene-labeled actin was prepared according to the
protocol of Brenner and Korn (6).
We determined the dissociation constant for the
-actin-profilin
interaction in the presence of gelsolin-capped filaments
as described
by Pantaloni and Carlier (
34). Time course experiments
were
carried out with 10 µM
-actin (10% pyrene labeled) and 5
µM
profilin using a Hitachi F-4500 spectrophotometer. The excitation
and
emission wavelengths were 365 and 388 nm, respectively. Polymerization
was started by adding MgCl
2 and KCl (to final
concentrations of
2 and 100 mM, respectively) to the preincubated
-actin-profilin
sample in G buffer (5 mM Tris-HCl [pH 7.7], 0.2 mM ATP, 0.2 mM
dithiothreitol, 0.1 mM CaCl
2).
The affinity of the profilins for polyproline was assayed with surface
plasmon resonance (BIACORE X). A VASP-derived peptide
[biotin-CGPPPPPGPPPPPGPPPPPGL-OH,
(GP
5)
3] was coupled to the
streptavidin-coated
sensor chip, and different concentrations of
profilin were passed
over the chip. Response units were measured for
each concentration.
The concentration at which half of the maximal
response (
Rmax/2)
is obtained is a measure of
affinity (
21).
We carried out PIP
2 binding as described earlier (
18,
26). Briefly, 5 µM profilin was incubated for 30 min on ice
with
different concentrations of PIP
2 micelles as indicated
in the
legend to Fig.
6. Subsequently, the sample was applied to a
Millipore
filter with a molecular weight cutoff of 30,000 and
centrifuged
for 1 min at 4°C. The flowthrough was analyzed by sodium
dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Western blotting and in situ hybridization on tissue
sections.
Dissected organs were homogenized in
radioimmunoprecipitation assay buffer containing 50 mM Tris (pH 8.0),
150 mM NaCl, 1% (vol/vol) Triton X-100, 0.5% (wt/vol) deoxycholate,
0.1% (wt/vol) SDS, 1 µM pepstatin, 1 mM phenylmethylsulfonyl
fluoride, 0.3 µM aprotinin, 1 µM leupeptin, 5 µM E-64, 1 mM EDTA,
1 mM sodium vanadate, 50 mM sodium fluoride, 2 mM levamisole, and 30 mM
sodium pyrophosphate, and homogenates were centrifuged for 30 min at
100,000 × g. Protein concentrations were determined
using the bicinchoninic acid assay (Pierce). Sixty-five micrograms of
total protein of each lysate was loaded on SDS-15% polyacrylamide
gels. SDS-PAGE and Western blotting were done by standard techniques.
The polyclonal antibodies used are G117 for profilin I and G124 for
profilin II and a goat anti-rabbit horseradish peroxidase conjugate as
secondary antibody. The signal was developed with the Renaissance
chemiluminescence reagent (Dupont-NEN). Concentration series of pure
profilin I and profilin IIa (indicated in Fig. 7) were used to
determine the relative amount of each profilin in the different
lysates, by comparing the signal intensities of the Western blots.
In situ hybridization of sections (
3) was performed with
digoxigenin-dUTP (Boehringer Mannheim)-labeled antisense or sense
riboprobes specific for the 3' untranslated regions of mouse profilin
I
(
49) and profilin II. For whole-mount in situ hybridization,
probes were prepared by in vitro transcription of linearized template
DNA using digoxigenin-labeled nucleotides. We used the mouse EST
sequences coding for either profilin I or profilin II (
AA871094 or
AA032658, respectively) as DNA templates. In situ hybridization
was
done as described previously (
19).
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RESULTS |
Comparing EST sequences leads to the identification of two profilin
II isoforms.
The conflicting data reported for bovine brain and
recombinant human profilin II were attributed to the use of profilin II proteins from different species. In a search for the mouse and rat
profilin II cDNAs, we screened mouse and rat EST databases (National
Center for Biotechnology Information) using amino acid sequences from
both human and bovine profilin II. We found several EST sequences that
are very similar to the bovine profilin II isoform. Sequencing mouse
EST AA032658 identified a 1,762-bp cDNA sequence containing a
25-bp-long 5' untranslated region, an open reading frame of 423 bp
ending with a TAG stop codon, and a 1,314-bp-long 3' untranslated
region. The open reading frame encodes a putative mouse profilin II of
140 amino acids, which is identical to bovine profilin II (Fig.
1). Interestingly, embedded in the 3'
region was an additional short open reading frame coding for a
polypeptide which showed 81% identity to the corresponding COOH-terminal part of human profilin II (pfn2, GenBank accession no.
NM_002628) (20) and only 59% to that of bovine profilin II
(25). The human profilin EST HS101130 (Fig. 1) is similar to
the mouse EST identified above containing potential coding information
for two different COOH termini.
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FIG. 1.
Alignment of the sequences of the various profilin
isoforms and translated cDNAs and ESTs from humans, rats, and mice. The
sequences are human (Hum) profilin I (pfn1; GenBank accession no.
NM_005022) (24), human profilin IIa (this work; accession
no. AF228738), human profilin IIb (pfn2; accession no. NM_002628) (this
work and reference 20), the translated Rat IIa+b
long transcript (RatIIa, accession no. AF228736, and Rat IIa+b,
accession no. AF228737), and MouseIIa+b (this work) (also derived from
EST AA032658). HS101130 is from human cells, and AA942886 is a
combination of two overlapping rat ESTs (AA942886 and H32106).
Profilins IIa and IIb differ only in the underlined region; residues
different in the IIb sequences are shaded. An asterisk denotes a stop
codon, X denotes an unidentified residue, and a dash denotes a gap; the
arrow indicates where the sequences diverge as a result of alternative
splicing (also Fig. 3).
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These results suggest the possible generation of two profilin II
isoforms by alternative splicing. We will refer to these
forms as
profilin IIa, which is the homologue of the protein purified
from
bovine brain (
25), and profilin IIb, which is similar to
the
human protein described by Honoré et al. (
20).
To verify the presence of alternatively spliced transcripts within the
same organism, we performed a series of PCRs on cDNA
libraries from
various human, rat, and mouse tissues using specific
primers for the
respective isoforms (for primers, see Table
1).
We cloned and sequenced
several of these transcripts, including
human and rat profilin IIa and
human and mouse profilin IIb (Fig.
1). The human profilin IIb
transcript corresponds exactly to the
human clone described earlier by
Honoré et al. (
20) and shows
95% identity with the
mouse homologue that is six amino acids
longer (146 amino acids versus
140 amino
acids).
In addition, using the profilin IIb primers we consistently amplified
from all human, rat (data not shown), and mouse (Fig.
2A) tissues examined a longer transcript.
These longer forms have
sequences very similar to those of the mouse
EST (
AA032658)
described above and contain profilin IIa coding
information (sequences
indicated as IIa+b in Fig.
1). Translation of
the mRNA from which
this PCR product is derived will result in the
profilin IIa isoform.
Interestingly, the PCR product for profilin IIb
is present in
only some mouse tissues, i.e., liver, kidney, and skin
(additionally,
it was present in human placenta [data not shown];
this tissue
was not probed in the mouse). This indicates that formation
of
the profilin IIb transcript is regulated in a tissue-dependent
manner. Note that the profilin IIb message is absent from the
brain
(see also below). Based on these quantitative RT-PCR experiments,
within a given tissue, we estimate that the mouse profilin IIa
message
in liver and skin is 7- to 8-fold, and in kidney 20-fold,
more abundant
than the profilin IIb message.
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FIG. 2.
Profilin IIa and IIb transcripts in mouse tissues. (A)
Quantitative RT-PCR on mRNA isolated from the indicated mouse tissues.
The forward and reverse primers used are E1Af and E4r, respectively
(see Table 1 for primer sequences and panel B for location). The short
PCR product (approximately 690 bp) is an amplification of the profilin
IIb mRNA. The longer transcript of approximately 960 bp contains coding
information for profilin IIa, a noncoding region, and information for
the C-terminal region of profilin IIb (also Fig. 1). -Actin
amplification was used as the control. (B) RT-PCR analysis of
alternative spliced transcripts of the mouse profilin II gene (lower
panel). First-strand cDNAs prepared from brain (b) and kidney (k) were
subjected to PCR using primer combinations E1Af and E3r (lanes 1), E1Af
and E4r (lanes 2), E1Bf and E3r (lanes 3), and E1Bf and E4r (lanes 4).
Primer sequences are listed in Table 1, and their locations are
indicated in the drawing. Profilin II transcripts spliced to exon 3 (corresponding to mRNA-a or -c [Fig. 4]) were detectable as strong
bands in both brain and kidney. Transcripts spliced to exon 4 (corresponding to mRNA-b or -d and marked with asterisks) were found
weakly expressed in kidney. M, molecular size marker (numbers are in
base pairs).
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Additionally, we characterized the 5' end of mouse profilin II
transcripts, using 5' RACE with adult mouse brain cDNA as template.
We
found two amplification products of different lengths (data
not shown).
The shorter product (216 bp) was identical to the
5' end of the
sequenced EST clone (
AA032658) and did not further
extend the 5'
untranslated region. Surprisingly, sequence analysis
of the longer
RACE clone (278 bp) revealed the existence of a
novel transcript
in which the 5' untranslated region and the nucleotides
encoding the
first 44 amino acids of profilin II were replaced
by a new sequence of
214 bp (see
below).
The exon-intron organization of the mouse profilin II gene gives
rise to at least four alternatively spliced messages.
To obtain
the genomic sequence of the mouse profilin II gene, we screened a mouse
P1-derived artificial chromosome library with an 833-bp-long
NcoI-PstI fragment derived from the EST
clone AA032658. We isolated one clone, digested it with
BamHI and EcoRI, and subcloned fragments
hybridizing with the cDNA probe into pBSII KS(+). Sequence analysis of
the plasmid inserts showed that the mouse profilin II gene spans over
7.3 kb including 1.6 kb of 5'- and 1 kb of 3'-flanking regions (Fig.
3 and 4).
Taking into account the mouse ESTs and RACE sequences described above, we predict five exons, flanked by consensus splice signals (Fig. 4C).
This could theoretically result in four types of mRNA (Fig. 4B). The
first type (represented by mRNA-a), spliced from exons 1A, 2, and 3, encodes profilin IIa. mRNA-b contains the sequence of exons 1A, 2, and
4 and codes for profilin IIb. mRNA-c and -d begin with exon 1B followed
by exons 2 and 3 and exons 2 and 4, respectively. The nucleotide
sequence of exon 1B is identical to the one of the longer RACE clone
described above, strongly suggesting that the splice products mRNA-c
and -d do exist.
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FIG. 3.
Nucleotide sequence of the mouse profilin II
gene (GenBank accession no. AF237680). Exons 1A, 1B, 2, and 3 are
boxed. Exon 4 is located inside exon 3 and is bordered by dashed lines.
Coding sequences of exons and corresponding amino acid residues are in
capital letters. Noncoding sequences of exons, intron sequences, and
the 5'- and 3'-flanking sequences are in lowercase. Numbering (left) of
the nucleotides includes intron sequences. Intron A is between exon 1A
and exon 2, intron B is between exon 1b and exon 2, intron C is between
exon 2 and exon 3, and intron D is between exon 2 and exon 4. The
potential transcription initiation nucleotide for exon 1A is in
boldface. The putative translation initiation codons and the
corresponding methionines in exon 2 are in boldface. The two consensus
polyadenylation signals are in boldface and underlined.
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FIG. 4.
Exon-intron organization of the mouse profilin II gene
and alternatively spliced transcripts. (A) The mouse profilin II
10,000-bp genomic sequence is shown as a thin horizontal line. Exons
(E) are indicated by boxes. Filled boxes represent coding sequences,
and open boxes represent noncoding sequences. Note that exon 4 (E4) is
located in the 3' untranslated region of exon 3 (E3). For comparison,
the human profilin I gene structure (sequence in AC004771.1) is also
given. (B) A diagram showing the four alternatively spliced profilin II
transcripts (mRNA-a to -d). mRNA-a and -b code for profilin IIa and
profilin IIb, respectively. The initiation codon ATG in exon 1A is
indicated. mRNA-c and -d start with exon 1B, are spliced to exon 2, and
then are spliced to exon 3 or exon 4, respectively. A putative
translation initiation codon (ATG) in exon 2 is shown. (C) Information
on sequences of splice donor and acceptor sites and intron and exon
length; for the location of the introns, see Fig. 3.
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To confirm the existence of mRNA-c and -d, we performed RT-PCR with
forward primers from exon 1B and reverse primers from
exons 3 and 4 (Table
1). The results were unambiguous (lanes
3 and 4 in Fig.
2B). In
brain and kidney tissues, a cDNA corresponding
to mRNA-c is present,
whereas one corresponding to mRNA-d (lacking
exon 3) is present in
kidney tissue. Comparing the ethidium bromide
staining intensities of
the amplified fragments revealed that
transcripts without sequences of
exon 3 are present in much lower
amounts than those having exon 3 sequences (Fig.
2B). Nevertheless,
our data indicate that four
different mRNAs can be produced from
the profilin II
gene.
mRNA-c and -d both contain an open reading frame starting in exon 2. At
present, we have no evidence that these transcripts
result in
functional proteins, but we note that the flanking region
of the second
in-frame ATG (nucleotide positions 3963 to 3965
[Fig.
3]) matches the
consensus sequence for translation initiation
sites (
23).
The proteins predicted from mRNA-c and -d are 55
and 61 amino acids
long and have calculated molecular weights
of 5,905 and 6,611,
respectively.
Profilins IIa and IIb have different biochemical properties.
To compare the profilin II isoforms biochemically, we expressed the
recombinant rat IIa and human IIb proteins in E. coli and purified them by poly-L-proline affinity chromatography
(see Materials and Methods). Bovine profilin IIa and recombinant human profilin I were used as a reference in all experiments.
We determined the dissociation constant for the
-actin-profilin
interaction for the different profilin isoforms using a classical
sequestration assay with capped filaments (
34). All
recombinant
forms have similar
Kd values for
-actin: human profilin I, 0.4
µM; human profilin IIb, 0.6 µM;
rat profilin IIa, 1 µM (Table
2). These
are consistent with values reported previously for
the nonrecombinant
forms of profilins I and IIa (
25,
34).
In addition, in a
time course polymerization experiment we observed
no major differences
between the different profilins (Fig.
5),
i.e., recombinant human profilins I and IIb and rat profilin IIa
decreased the rate of
-actin polymerization and reduced the total
amount of F-actin to a similar extent as did profilin IIa purified
from
bovine brain.
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FIG. 5.
The profilin isoforms have similar effects on actin
polymerization. We monitored the change in pyrene fluorescence
(relative fluorescence [R.F.]) after induction of polymerization with
2 mM MgCl2 and 0.1 M KCl of 10 µM actin alone ( ) or in
the presence of 5 µM human profilin I ( ), human profilin IIb
( ), rat profilin IIa ( ), or bovine profilin IIa (×).
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We used surface plasmon resonance technology to compare the affinities
of the profilin isoforms for peptide (GP
5)
3,
corresponding
to the proline-rich sequence in VASP. As a measure for
affinity,
we determined the molar concentration of profilin needed to
reach
half of the maximal response (for details of the procedure, see
reference
21). This concentration for rat profilin
IIa is 0.3
to 0.5 µM (Table
2), in agreement with previous data
obtained
for bovine profilin IIa, where we found a concentration of 0.5
µM with a stoichiometry of two profilin molecules for one peptide
(
21). The high-affinity interaction is due to cooperative
binding
of the two profilin molecules to one peptide. For recombinant
human profilin IIb and profilin I, we consistently found a low
response
(data not shown), in agreement with previous observations
for bovine
profilin I and different types of poly-
L-proline binding
studies (
26,
37). The fact that the response unit values
were
low, even at the highest concentrations tested, indicates that
the
affinity of isoforms I and IIb for proline-rich sequences
is much lower
than the affinity of profilin
IIa.
We assayed PIP
2 binding in a microfiltration experiment.
The flowthrough, representing the nonbound part of profilin, was
analyzed by SDS-PAGE, and the amount of protein was quantified
(Fig.
6, inset). From these data, the
percentage of bound profilin
can be calculated (Fig.
6). Less profilin
IIa than profilin I
was associated with the PIP
2 micelles,
and binding of profilin
IIb to PIP
2 was even more reduced.
The results obtained for recombinant
profilin IIa are similar to those
described previously for bovine
profilin IIa (
26). Thus,
both profilin II forms have a reduced
affinity for PIP
2
compared to that of profilin I (Table
2).
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FIG. 6.
Profilin isoforms have different affinities for
PIP2. The bars represent the percentages of bound profilin
at the PIP2 concentrations indicated. These values were
calculated from the amounts of nonbound profilin found in the
flowthrough after microfiltration. Values for human profilin I (grey
bars), rat profilin IIa (white bars), and human profilin IIb (hatched
bars) are shown. The average of two experiments is shown. The inset
shows SDS-polyacrylamide gels of the flowthrough fractions.
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Profilin IIa is mainly expressed in neural tissues.
Western
blotting experiments were performed with polyclonal rabbit antibodies
G117 and G124, raised against bovine profilin I and bovine profilin
IIa, respectively. G124 does not discriminate between profilins IIa and
IIb, but given our RT-PCR results (see above and Fig. 8C), we probed
profilin IIa expression in brain tissue. G117 and G124 displayed no or
very weak cross-reactivity with the other isoform (Fig.
7). Based on this concentration series, we determined the amounts of profilin I or IIa in the different tissues
examined.
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FIG. 7.
Antibodies G117 and G124 distinguish between profilin I
and profilin II on Western blots. Decreasing amounts (as indicated) of
human profilin I and bovine profilin IIa were used to test the
specificity of the antibodies. Based on the intensities on these
Western blots, we determined relative amounts of profilin in the
lysates (Fig. 8A, B, and D) by performing the Western blottings
simultaneously.
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Profilin I was found in relatively high concentrations in all tissues
tested except for skeletal muscle and heart (Fig.
8A).
In contrast, using this approach,
profilin IIa was detectable
only in brain (reference
50 and data not shown). Since both
profilin I and
profilin IIa are expressed in the brain, we probed
different adult
brain regions and found that both isoforms are
present in all regions
of the brain tested. Profilin IIa is expressed
weakly in the striatum
and the olfactory bulb and more strongly
in the cerebellum, pons and
medulla, midbrain, cortex, and hippocampus
(Fig.
8B).
View larger version (58K):
[in this window]
[in a new window]
|
FIG. 8.
Western blot analysis and RT-PCR of mouse tissues and
brain regions. (A) Adult mouse organs probed with polyclonal
anti-profilin I antibody G117. sk., skeletal. (B) Adult mouse brain
regions: cerebellum (cereb.), pons/medulla (pons/meds), midbrain
(midbr.), cortex, striatum, hippocampus (hippoc.), and olfactory bulb
(olf.bulb) probed with polyclonal antibodies G117 and G124 against
profilin I and profilin IIa, respectively. (C) Quantitative RT-PCR,
using primers E1Af and E4r, of profilin messages at the indicated
stages of mouse development. Only the long transcript encoding profilin
IIa is observed. Lane M contains size markers (1,018-bp upper marker
and 517-bp lower marker). -Actin amplification was used as the
control. (D) Developmental stages of mouse brain prepared from total
brains of E11 to E16 embryos and different regions of E18 and P1
brains. H, hippocampus; S, striatum; C/O, cortex and olfactory bulb; M,
midbrain; C, cortex; CPM, cerebellum, pons, and medulla.
|
|
We also investigated profilin isoform expression during
mouse development. RT-PCR, with profilin IIb-specific primers
(amplifying
the profilin IIb message and/or the long transcript coding
for
profilin IIa [see above]), of RNA isolated from various mouse
embryonic stages revealed the presence of solely the long transcript,
indicating that only profilin IIa is significantly expressed (Fig.
8C).
We next addressed differences in profilin I and profilin
IIa expression
during brain development by Western blotting. Embryonic
heads at stages
E11 to E16 and E18 and P1 brain regions were dissected
(Fig.
8D). The
expression level for profilin I is constant during
embryogenesis (0.004 to 0.008% of total protein) and does not
increase after birth, while
profilin IIa expression is clearly
upregulated from stage E14 on and
increases until after birth
(0.004 to 0.075% of total protein). The
antibodies hardly detected
profilin IIa in earlier stages of
development.
To analyze profilin IIa expression at these earlier stages, we
performed in situ hybridization on mouse E8.5 to E11.5 whole-mount
embryos (Fig.
9A to H). Whereas profilin
I mRNA was found in most
parts of the embryo, even at the early stage
E8.5, no profilin
IIa mRNA could be detected at stages E8.5 and E9.5
(data not shown),
although the message could be amplified by RT-PCR
(Fig.
8C). For
E10.5 embryos, profilin I was seen in the
forebrain and midbrain,
the pharyngeal and branchial arches, and
the four limb buds (Fig.
9A and B). AT E11.5, profilin I expression was
prominent in most
fetal organs, including heart, liver, and kidney
(Fig.
9D). In
contrast, profilin IIa expression was limited to the
developing
brain and the neural tube (Fig.
9E to H). Profilins I and
IIa
stained different structures on the dorsal sides of the embryos.
The somites expressed profilin I (Fig.
9C), whereas profilin IIa
was
expressed in the rhombic lip and the lateral region of the
neural tube
(Fig.
9G).
View larger version (144K):
[in this window]
[in a new window]
|
FIG. 9.
In situ hybridization of whole-mount mouse embryos and
adult mouse brain sections. (A to H) Expression patterns of profilin I
(A to D) and profilin II (E to H) during early embryonic development.
(A, B, E, and F) Lateral view of E10.5 embryos. Profilin I (A and B) is
found in the midbrain (Mb), forebrain (Fb), branchial arches (Ba),
pharyngeal arches (Pa), forelimb bud (Flb), and hind limb bud (Hlb).
Profilin II expression (E and F) was detected in the entire brain
region and the neural tube. (C and G) Dorsal view of E10.5 embryos
shows profilin I expression in the somites (S) and profilin II in the
rhombic lip (Rl) and the lateral neural tube (Lnt). (D and H) Lateral
views of E11.5 embryos showing profilin I expression (D) in the
olfactory region (Or), tongue epithelium (Te), heart (H), atrium (A),
liver (L), kidney (K), somites, and hind limb bud and profilin II
expression (H) in the developing brain and the neural tube (Nt). (I to
P) Overlapping expression of profilin I and profilin II transcripts in
adult mouse brain. Coronal (I to L) or sagittal (M to P) paraffin
sections were hybridized with digoxigenin-labeled antisense riboprobes
specific for profilin I (I, K, M, and O) or profilin II (J, L, N, and
P). (I and J) Profilins I and II are expressed in the dentate gyrus
(DG) and the CA regions of the hippocampus. (K and L) Strong expression
of profilins was detected in all layers of the cortex. (M and N)
Intensive signals were observed in the Purkinje cells (arrows) of
cerebellum. Cells in both the molecular (ML) and granular (GL) layers
were positive for profilins I and II. (O and P) Main olfactory bulb;
strong staining was seen in the mitral cell layer (MCL).
|
|
We also employed in situ hybridization to compare the expression
patterns of profilins I and IIa on tissue sections from 2-month-old
mouse brains. In agreement with the Western blotting data, both
profilin I and profilin IIa were detected in various regions of
the
central nervous system including the cerebrum, cerebellum,
and
olfactory bulb (Fig.
9I to P). Identical expression patterns
of
profilins were observed in the dentate gyrus and the cornus
ammonis
(CA) regions, in the hippocampus (Fig.
9I and J), and
in the cortical
layers of the forebrain (Fig.
9K and L). In the
cerebellum, Purkinje
cells were highly positive for both profilin
I and profilin IIa (Fig.
9M and N, arrows). Coexpression of profilin
I and profilin IIa was also
detected in the cells of the molecular
and the granular layers (Fig.
9M
and N). In the olfactory bulb,
the strongest signals were observed in
the granule cells of the
mitral cell layer (Fig.
9O and
P).
| |
DISCUSSION |
The five exons in the profilin II gene are used to form four
different messages.
In this paper, we report the profilin II gene
sequence and deduce the exon-intron structure. At least four different
mRNAs are generated from this gene by alternative splicing. Two of
these are translated to different profilin II isoforms with distinct biochemical properties. Thus, our results clarify conflicting observations previously reported for bovine (25, 26) and
human (14) profilin II which were due to the use of two
different isoforms, designated here profilins IIa and IIb,
respectively. The two other messages potentially code for shorter
proteins lacking the NH2-terminal parts of profilins IIa
and IIb. These fragments await further characterization, but we note
that these polypeptides, if they adopt a three-dimensional structure
similar to that in profilin, would possess a large hydrophobic surface,
suggesting that they are associated with another protein or are
membrane bound. In addition, these truncated forms lack some of the key residues involved in poly-L-proline binding (e.g., W3, Y6,
and W31; numbering is without the initiator methionine that is
posttranslationally removed) (4, 5, 30, 31) and actin
binding (e.g., F59, V60, S71, R74, E82, and T84)
(39; also discussed in reference 33), suggesting that these truncated forms do not
bind these two ligands.
Profilins IIa and IIb are differentially expressed.
We
observed ubiquitous expression of profilin I using Western blotting and
RT-PCR, suggesting that it is the major form in almost all tissues. The
profilin IIa message can also be detected in most tissues using a
sensitive RT-PCR. The protein is, however, only abundantly expressed in
the brain, where it is the major isoform in some regions, in which it
is present at two- to fivefold-higher levels than those of profilin I. In the adult brain, we did not observe regions expressing either
profilin IIa or profilin I separately. The two profilins are relatively
highly expressed in the cortical layers of the forebrain, in the CA
regions, and in the dentate gyrus, where Mena is also strongly
expressed (32). The occurrence of profilin IIb is rare. It
is not expressed during embryogenesis, but the mRNA is present in mouse
kidney, skin, and liver. This restricted expression pattern indicates
that generation of this splice variant is tightly regulated.
The expression pattern of profilin IIa suggests a neuronal
function.
During all stages of brain development, profilin I
expression is constant. Profilin IIa mRNA can first be detected at E9.5 to E10. The protein level steadily increases until after birth. Strong
profilin IIa expression is restricted to brain structures, which
suggests a specific function for this isoform in central nervous system
and neural tube development. The neural tube closure defects of
Mena
/ profilin I/+ mice indicate an
important role of profilin in the migration of neuroepithelial cells
(27). Since profilin IIa expression starts after neural tube
closure, it cannot compensate for profilin I during this process.
The higher in vitro affinity of profilin IIa for proline-rich
peptides derived from VASP (
21,
26), EVL
(Ena/VASP-like
protein [
26a]), and Mena [and
probably Mena(+)] (A. Lambrechts,
V. Jonckheere, and C. Ampe,
unpublished data) suggests that profilin
IIA is a preferred partner of
Ena/VASP proteins. Interestingly,
expression of EVL in brain starts
around E15, and the neuron-specific
splice variant of Mena, Mena(+),
shows a maximum expression level
between E15 and P1 (
27),
which correlates with profilin IIa
expression. These data suggest a
function for profilin IIa in
modulating actin reorganization at
specific points during brain
development.
Additionally, profilin II may be involved in regulation of synaptic
vesicle trafficking, based on the profilin immunostaining
at
presynaptic sites and binding of profilin to several synaptic
proteins
(
12,
50). Aczonin, a protein containing a proline-rich
region and concentrated at active zones of presynaptic sides,
binds
more strongly to profilin IIa than to profilin I (
46).
Other
partners of profilin II in neuronal tissues are protein
isoforms
encoded by the SMN gene and responsible for spinal muscular
atrophy.
These candidate proteins for determining loss of motoneurons
were
reported to associate preferentially with profilin II (
15).
Currently, it is not known whether both isoforms of profilin II
or only
profilin IIa can associate with SMN
protein.
The three profilin isoforms have different biochemical
properties.
Profilins are small proteins, which bind multiple
ligands. Therefore, they are thought to act at the crossroads of
different signaling pathways toward the actin cytoskeleton
(41). The presence of two or three different isoforms with
different biochemical properties in a cell allows better fine-tuning of
signaling to the actin system. For each of the three profilin isoforms,
we observed similar Kds for -actin, but
obviously the natural partners of profilin in nonmuscle cells are -
and 
-actin. The previously reported lower affinity of profilin
IIb for -actin (14) may arise from averaging
Kd values derived from different assays using capped and noncapped filaments. The similar affinities correlate well
with conservation of profilin residues in the interface with actin
(33, 39).
Although it is generally accepted that basic residues in proteins
mediate the interaction with PIP
2, their identity remains
elusive, as conflicting data were reported previously for mutated
profilins from invertebrates and mammals (
18,
42). Recent
studies have proposed that residues from the NH
2- and
COOH-terminal
helices are implicated in PIP
2 binding
(
5,
9,
10). The
latter result is consistent with our recent
data that suggest
a role for amino acids Arg135 and Arg136 of human
profilin I in
binding to PIP
2. Mutating Arg136 to Asp
results in reduced PIP
2 affinity (A. Lambrechts, V. Jonckheere, D. Dewitte, J. Vandekerckhove,
and C. Ampe, unpublished
results). Interestingly, profilin IIa
has this amino acid change,
which may explain why this form has
a reduced affinity for
PIP
2 compared to that of profilin I. Profilin
IIb
has, however, an arginine at position 136 but also an aspartic
acid
residue at position 138, which may cause the lower binding
affinity of
this
isoform.
The elucidation of two crystal structures with different
modes of binding of profilin I to proline-rich peptides (
30,
31)
complicates interpretation of the data on
(GP
5)
3 binding of the
various profilin
isoforms. It is clear that the high-affinity
binding that we observe
for a dimer of profilin IIa molecules
is cooperative (
21).
The reduced affinity of profilins I and
IIb may then be due to
inefficient dimer formation, i.e., the
profilins bind independently
from each other, consistent with
the lack of contact between the
profilin molecules in the profilin
I dimer
L-Pro10 crystal
structure (
30). Alternatively, data
from the profilin IIb
crystal structure (
33) suggest an aromatic
extension of the
binding pocket by an additional stacking interaction
and a hydrogen
bond with poly-
L-proline by tyrosine 29 (serine
in profilin
I) which is found in both profilin II isoforms. The
lower affinity of
profilin IIb for proline-rich sequences (this
study and reference
14) may then be due to negative effects
exerted by
its C-terminal part. We note that the aromatic character
of the
C-terminal residue, known to be important in contacting
prolines
(
4,
5,
33), is conserved in profilin IIa but
not in profilin
IIb.
Our data indicate that each of the three isoforms has a different
combination of binding affinities to the ligands PIP
2 and
(GP
5)
3, which serve as models for
polyphosphoinositol lipids and
proteins from the Ena/VASP family,
respectively. Profilins may
use these different ligand binding
properties for modulating actin
polymerization in a different manner.
Indeed, the effect on actin
interaction with these ligands is very
different. PIP
2 inhibits
the interaction with actin
(
28), whereas a ternary complex can
be formed among
poly-
L-proline, profilin (I or II), and actin
(
21,
25,
36). In the case of profilin IIa, dimerization
on
(GP
5)
3 results in an enhanced desequestration
of actin from
the pool bound to thymosin
4 and in increased actin
polymerization
(
21). In addition to a modulatory role, these
ligands may recruit
either isoform to different subcellular locations
depending on
the cellular context, the available ligands, and the
concentration
of the profilin
isoforms.
| |
ACKNOWLEDGMENTS |
Anja Lambrechts and Attila Braun contributed equally to this paper.
We thank F. Van Roy for use of the DNA sequencing facility for some of
the profilin clones. We acknowledge Leen Van Troys for critically
reading the manuscript and Griet Vandeweghe and Daniel Broekaert for
assistance with additional experiments. Part of this work was carried
out in the laboratory of Frank Gertler (Massachusetts Institute of
Technology, Cambridge), and we acknowledge his contribution.
A.L. was the recipient of a travel grant from the Fund for Scientific
Research-Flanders (FWO-Vlaanderen, Belgium) to work at MIT. C.A. is a
research associate of the Fund for Scientific Research-Flanders. This
work was supported by grants G004497, G006096, and G022598 of the Fund
for Scientific Research-Flanders to C.A. and J.V.; a grant from the
"Geneeskundige Stichting Koningin Elisabeth" to C.A.; GOA grants
12051296 and 120C1797 to J.V.; and a grant of the Swedish Cancer
Foundation to R.F.
| |
ADDENDUM IN PROOF |
A paper describing the profilin II gene structure and the
alternative splice variants profilins IIa and IIb by Di Nardo et al. is
in press in J. Cell. Sci. The nomenclature used for the two profilin
isoforms is the same as in this paper.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Ghent University, B-9000 Ghent, Belgium. Phone: 32 9 2645306. Fax: 32 9 2645337. E-mail:
champ{at}gengenp.rug.ac.be.
| |
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Molecular and Cellular Biology, November 2000, p. 8209-8219, Vol. 20, No. 21
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
