From the We have cloned and sequenced a 9.4-kilobase
cDNA specifying a new 280-kDa protein interacting with the
cytoplasmic tail of glycoprotein (Gp) Ib Glycoprotein (Gp)1
Ib In platelets, the GpIb complex is tightly bound to the actin
cytoskeleton via an interaction of GpIb Partial sequence of two other ABP-280-related entities from human
sources has been reported. Two partial cDNA fragments of one
species, termed actin-binding protein-like protein (ABPL), were cloned
by PCR and mapped to chromosome 7; full-length cDNA has not been
reported. A second species, termed truncated actin-binding protein
(TABP) in one publication (9) and filamin homolog-1 in another (10),
has been reported. The TABP open reading frame predicts a protein of
only 195 residues, whereas only a 291-amino acid sequence has been
reported for filamin homolog-1. However, both sequences are homologous
to the COOH-terminal region of ABP-280.
Here, we report the molecular cloning and full sequence of a new human
homolog of chicken filamin, which, like ABP-280, associates with the
cytoplasmic tail of GpIb Two-hybrid Library Screening and Full-length cDNA
Cloning--
A DNA fragment coding the cytoplasmic tail of GpIb Cardeza Foundation for Hematologic Research,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and showing considerable
homology to actin-binding protein 280 (ABP-280) and chicken retinal
filamin. We term this protein human
-filamin. The gene for
-filamin localizes to chromosome 3p14.3-p21.1.
-Filamin mRNA
expression was observed in many tissues and in cultured human umbilical
vein endothelial cells (HUVECs); only minimal expression was detected
in platelets and the megakaryocytic cell line CHRF-288. Like ABP-280,
-filamin contains an NH2-terminal actin-binding
domain, a backbone of 24 tandem repeats, and two "hinge" regions. A
polyclonal antibody to the unique
-filamin first hinge sequence
identifies a strong 280-kDa band in HUVECs but only a weak band in
platelets, and stains normal human endothelial cells in culture and
in situ. We have confirmed the interaction of
-filamin
and GpIb
in platelet and HUVEC lysates. In addition, using
two-hybrid analysis with deletion mutants, we have localized the
binding domain for GpIb
in
-filamin to residues 1862-2148, an
area homologous to the GpIb
binding domain in ABP-280.
-Filamin is a new member of the filamin family that may have significance for
GpIb
function in endothelial cells and platelets.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, GpIb
, GpIX, and GpV are all members of the leucine-rich
glycoprotein superfamily and form a complex in the platelet membrane
(1). All four polypeptides of the GpIb complex are also present in
human endothelial cells (2). The platelet GpIb complex mediates the
adherence of platelets at the site of vascular injury through the
binding of GpIb
to subendothelial von Willebrand factor (vWF).
with actin-binding protein 280 (ABP-280) (3-5); it can be presumed (but has not been shown) that
a similar interaction occurs in endothelial cells (ECs). ABP-280
cDNA has been cloned from a HUVEC cDNA library (6) and mapped
to the X chromosome (7, 8). ABP-280 has sometimes been referred to as
human non-muscle filamin.
. It is present in only small amounts in
platelets, but in substantial quantities in ECs. We term this new
protein human
-filamin, reserving the term
-filamin for
ABP-280.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
between residues 1633 and 1976 (11) was amplified from human genomic DNA (because the GpIb
gene has no introns in this area),
directionally cloned in-frame into the yeast expression vector pAS2-1
(CLONTECH), and confirmed by sequencing. This
vector expresses a fusion protein consisting of the cytoplasmic tail of
GpIb
and the yeast GAL4 DNA-binding domain.
tail vector and the library vectors.
Transformants were plated on
SD/Trp
/Leu
/His
plates, and
incubated at 30 °C until colonies appeared.
-Galactosidase activity of each colony was determined qualitatively in a filter-lift assay. In order to exclude false positives, positive clones were re-introduced into yeast together with either the GpIb
bait vector, the pAS2-1 original vector or pLAM5', a vector encoding a human lamin
cDNA in the two-hybrid DNA-binding domain vector pGBT9. To obtain
the remaining 5' cDNA, a human placenta 5'-STRETCH cDNA library
in
gt10 (CLONTECH) was screened as described
under "Results."
Sequence Analysis--
We used the BLAST computer program to
search the GenBank data base for expressed sequence tags (ESTs) and
sequence-tagged sites (STSs) containing sequences identical to those in
-filamin cDNA. The UNIGENE program was then used to group ESTs
and STSs.
Northern Blot Hybridization and RT-PCR--
A human multiple
tissue blot was purchased from CLONTECH, and a blot
of HUVECs and CHRF-288 cell poly(A)+ RNA was kindly
supplied by Dr. Barbara Konkle, Cardeza Foundation, Jefferson Medical
College. -Filamin mRNA expression was detected with a ~2.6-kb
probe from the 5' end of cDNA clone THC-106 (Fig. 1). ABP-280
mRNA expression was detected with a ~2.7-kb (nt 5605-3' end)
ABP-280 cDNA clone isolated from the bone marrow library. To
confirm the quality of the blots, we used
-actin as a probe.
Antibody Production and Western Blotting--
Because the first
"hinge" region in -filamin differs completely from that in
ABP-280 (Fig. 4), we synthesized a peptide (TDGEVTAVEEAPVNACPPG) corresponding to that sequence and coupled it to activated keyhole limpet hemocyanin or to activated bovine serum albumin (BSA) (Pierce). The keyhole limpet hemocyanin peptide was used as immunogen. Rabbits were injected every 2-3 weeks, and serum titers were measured on the
BSA-peptide-coated ELISA plates. IgG fractions were isolated from serum
using protein G-Superose (Amersham Pharmacia Biotech) and
affinity-purified on BSA-peptide Sepharose columns.
Immunohistochemistry--
HUVECs were incubated overnight at
4 °C with a mAb to ABP-280 (mAb-4, kindly supplied by Dr. John
Hartwig) or to vWF (Dako, Carpinteria, CA), or an irrelevant control
mouse IgG1 antibody (Dako). Bound mouse IgG was detected using a
biotinylated horse anti-mouse polyclonal antibody (Vector Labs,
Burlingame, CA) followed by avidin conjugated to Texas Red (Leinco
Technologies, Inc., Ballwin, MO). After appropriate washing the cells
were incubated for 1 h at room temperature with either rabbit
polyclonal antibody to the -filamin first hinge, rabbit polyclonal
antibody to vWF (Biogenex, San Ramon, CA), or rabbit polyclonal control
immunoglobulins (Biogenex). Bound rabbit IgG was detected using
fluorescein-conjugated goat anti-rabbit IgG (Leinco). Stained HUVECs
were visualized by confocal laser scanning microscopy.
Interaction of -Filamin and GpIb
in Whole Cell
Lysates--
Microtiter wells were coated with 100 µl of a 10 µg/ml solution in 0.1 M
NaHCO3/Na2CO3, pH 9.6, of either
purified anti-GpIb
mAb Ib1 (12) (kindly supplied by Dr. Zaverio
Ruggeri, Scripps Research Institute, La Jolla, CA) or normal mouse IgG
(Pel-Freez, Rogers, AR). After washing three times with PBS, the wells
were blocked with 3% BSA-PBS. Platelets and HUVECs were lysed with lysis buffer at concentrations of 1.5 × 109/ml and
5 × 106/ml, respectively, and centrifuged at
100,000 × g for 90 min, in order to remove
cytoskeleton actin filaments (3). Wells were incubated with serial
dilutions of either platelet or HUVEC supernatant for 4 h at room
temperature and then with fresh supernatant overnight at 4 °C. The
wells were washed six times and incubated for 1 h with either 100 µl of a 10 µg/ml solution in 3% BSA-PBS of anti-GpIb
polyclonal
IgG (13), affinity-purified polyclonal anti-
-filamin IgG, or
pre-immune rabbit IgG. The wells were then washed six times with PBS,
incubated with 100 µl of phosphatase-labeled goat anti-rabbit IgG
(Kirkegaard and Perry, Gaithersburg, MD), washed six times, and
developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue
tetrazolium phosphatase substrate (Kirkegaard and Perry). Experiments
were done in duplicate.
Identification of the -Filamin Domain Binding to the
Cytoplasmic Tail of GpIb
--
Deletion mutants in the vector pACT2
were made using cloning sites of the vector and internal restriction
sites of
-filamin (ApaI at nt 5708, NcoI at nt
6571 and nt 7462, and EcoRI at nt 7341) (Fig. 10). Because
all inserts except clone 1 lacked stop codons in their sequence,
expression of clones 2-6, which required use of vector stop codons,
resulted in products containing 10 additional amino acids. Each mutant
was co-transformed into yeast with the GpIb
bait vector and
processed for quantitation of
-galactosidase activity using a liquid
culture assay with
o-nitrophenyl-
-D-galactoside (Sigma) as
substrate.
-Galactosidase units were calculated as recommended by
the supplier (CLONTECH).
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RESULTS |
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Isolation of -Filamin cDNA--
Using as bait the
cytoplasmic tail of GpIb
, we first screened a human bone marrow
two-hybrid library. Of 4 × 106 clones screened, we
isolated six His+/LacZ+ clones, each with the
same size ~2.7-kb insert. The sequence of one of these clones was
identical to the sequence of ABP-280 (6). Because ABP-280 cDNA has
an EcoRI site within this sequence, we compared the cleavage
patterns of the remaining five clones with that of the sequenced clone,
and found them to be identical.
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|
Homology between -Filamin, CRF, and ABP-280--
-filamin
shows strong homology to CRF (15) and human ABP-280 (6) over its entire
sequence, with the exception of the first hinge region, and with other
actin-binding proteins, including spectrins,
-actinin and
dystrophins, in its amino terminus (residues 1-275).
-Filamin,
ABP-280, and CRF all contain 24 internally homologous repeats (Fig.
3);
-filamin and ABP-280 contain two hinge regions, whereas CRF contains only the more COOH-terminal of the
two hinge regions. Furthermore, although the second hinge regions of
-filamin and ABP-280 are 44% identical, the first hinge regions of
the two proteins are entirely dissimilar (Fig. 4). Excluding the first hinge region, the
amino acid sequence of
-filamin is 70% identical with ABP-280, but
83% identical with CRF, whereas ABP-280 is 66% identical with CRF.
The partial sequence of ABPL (the chromosome 7 homolog) is 68%
identical with CRF and resembles CRF in lacking a first hinge; the
available sequence does not extend sufficiently 3' to identify the
second hinge region (8).
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Chromosomal Localization of -Filamin--
During analysis of
-filamin sequence data, we observed that an STS marker, WI-8718, was
part of the gene. This marker has been linked to microsatellite marker
D3S1295 in 3p14.3-p21.1 by analysis of the GeneBridge 4 Radiation
Hybrid Panel at the Whitehead Institute.2 The
-filamin
gene maps between D3S1313 and D3S1295.
Tissue and Cellular mRNA Expression of
-Filamin--
Northern blot analysis revealed that
-filamin
(Fig. 5A), like ABP-280 (Fig.
5B), is constitutively expressed in many adult human
tissues. However, there appear to be some differences in the
tissue-specific expression of the two species (for example, kidney and
pancreas versus heart and lung). Two different-sized
-filamin mRNA transcripts were detected (Fig. 5A),
one somewhat larger than 9.5 kb and one somewhat smaller than 9.5 kb,
the latter almost identical in size to the ABP-280 transcript (Fig.
5B). These differences are most likely a result of the use
of two different poly(A) signal sequences, one located just beyond the
stop codon and one located 1.5 kb further 3' (data not shown). The
cDNA that was isolated from the two-hybrid library used the later
poly(A) signal sequence. However, we also found several EST clones
(H01996, W40252, W45289, W52463, AA056341, T67924) that use the earlier
signal.
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|
Detection of -Filamin Protein--
By Western blotting with the
antibody to
-filamin's unique first hinge, we detected substantial
amounts of 280-kDa protein in HUVECs, but only minimal amounts in
platelets (Fig. 7). This band was
completely eliminated by pre-incubation of the antibody with 10 µg/ml
first hinge peptide (data not shown). The lower molecular mass bands
observed were also seen in the absence of the specific hinge antibody
(data not shown). ABP-280 protein was expressed in substantial amounts
in both cell types. Thus it appears that in platelets ABP-280
predominates, whereas relatively similar amounts of the two proteins
are present in HUVECs.
|
Immunohistochemistry--
As shown in Fig.
8a, -filamin is detectable
in normal human dermal vessels; the vessel shown here is a venule. The
pattern is the same as that seen for GpIb
(data not shown). As shown in Fig. 8b, by confocal microscopy
-filamin in cultured HUVECs is
localized primarily on the cytoplasmic aspect of the cell and nuclear membranes, but also appears in a more linear distribution within the cytoplasm; ABP-280 shows a similar pattern (data not shown). In contrast, the punctate staining of von Willebrand factor is
shown in Fig. 8c.
|
Association between GpIb and
-Filamin--
We used an
antigen-capture ELISA assay to detect the association between GpIb
and
-filamin in 100,000 × g supernatants of Triton
extracts of platelets and HUVECs. The anti-GpIb
mAb Ib1 captured
GpIb
(Fig. 9, open bars)
from both platelet and HUVEC lysates in a dose-dependent
manner; the lesser amount captured from HUVECs is consistent with the
lower level of GpIb
protein expression in HUVECs than in platelets
(2). The anti-
-filamin polyclonal antibody detected
-filamin
associated with the GpIb
captured by mAb Ib1 (Fig. 9,
diagonally striped bars). The solid bars depict
controls using pre-immune rabbit IgG in place of the primary
antibodies. Because the antibodies used for the platelet and EC
supernatants were identical, we compared the absorbance ratios of
captured
-filamin to GpIb
for platelets and ECs. The
-filamin:GpIb
ratios for the 1:2 dilutions (after subtracting the
pre-immune rabbit IgG control) were 0.08 and 1.34 for platelets and
ECs, respectively, suggesting that a much larger fraction of EC GpIb
than platelet GpIb
is bound to
-filamin. When normal mouse IgG
was used for coating the ELISA wells, no
-filamin was captured (data
not shown).
|
Localization of the Domain of -Filamin Binding to the
Cytoplasmic Tail of GpIb
--
To determine the domain of
-filamin interacting with the cytoplasmic tail of GpIb
, we
performed two-hybrid analysis. Six constructs were prepared in pACT2,
as indicated in Fig. 10. Each construct
expressed a fusion protein consisting of the yeast GAL4-activation domain and one cDNA fragment. After co-transformation of each construct with the GpIb
bait vector, we analyzed expression of HIS3
and lacZ by growth selection and
-galactosidase assay, respectively. As shown in Fig. 10, fusion constructs 1, 2, and 3 were positive in
both assays, whereas construct 4, lacking residues 1862-2148, was
negative. These results indicate that the GpIb
binding domain in
-filamin is located within residues 1862-2148 (repeats 17-20). This GpIb
binding domain in ABP-280 has been reported to localize to
residues 1850-2136 (repeats 17-19) (16). Constructs 5 and 6 induced
HIS3 activity and construct 5 also induced a small amount of lacZ
activity in the filter assay; however, they did not induce lacZ
activity in the quantitative assay using
o-nitrophenyl-
-D-galactoside. Because X-gal
detection in the filter assay is ~106 times more
sensitive than o-nitrophenyl-
-D-galactoside,
it is still possible that the polypeptide sequence between 2406 and 2445 includes another weak binding site to GpIb
.
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DISCUSSION |
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The term "filamin" was introduced by Singer's laboratory (17)
to describe a 250-kDa protein isolated from chicken gizzard, normally
present as a dimer (18), that is capable of inducing actin
polymerization (19). Filamins have been isolated from a number of
organs and cell types from chicken, human and other species (20-24),
but most biochemical studies have been performed using filamins
isolated from chicken gizzard, rabbit macrophage or human uterus. In
1990, Gorlin et al. (3) reported the sequence of a human
endothelial cell cDNA specifying a protein which they termed
actin-binding protein 280, or ABP-280. This protein is probably
identical to the actin-binding protein purified from human uterus,
based on limited amino acid sequence (25) and on cross-reactivity with
monoclonal antibodies (6), and which exists as a 560-kDa dimer (6).
Because of its 280-kDa size, its ability to dimerize, and its ability
to induce actin polymerization, ABP-280 has often been referred to as
human non-muscle filamin, although it is present in smooth (uterine)
muscle (6, 25) and in skeletal muscle (Ref. 8, this paper, Fig. 5).
ABP-280 contains a 274-residue amino-terminal actin-binding domain,
followed by 24 internally homologous repeats among which are
intercalated two hinge regions, one of 32 residues between repeats
15-16 and one of 34 residues between repeats 23-24 (these figures are
based on our sequence alignment of ABP-280, -filamin, and CRF).
Gorlin et al. (6) localized the site of dimerization of
ABP-280 to repeat 24. Electron microscopic studies of human uterine
actin-binding protein by these investigators suggested that the first
hinge region might allow the ABP-280 dimer to form a "Y," with the
COOH-terminal halves of the protein forming a rigid dimeric structure
and the NH2-terminal halves, containing the actin-binding
domains, forming the separated ends of the Y. The two hinge regions
were found to be sites of calpain cleavage, resulting in the formation
of 185-, 90-, and 10-kDa fragments (6, 26).
In 1993, Barry and co-workers (15) cloned and sequenced chicken retinal filamin and found it to be highly homologous to ABP-280, with the major exception that the CRF sequence lacked the more NH2-terminal of the two hinge regions present in ABP-280 (15). If one assumes that CRF and chicken gizzard filamin are identical and that human ABP-280 and rabbit actin-binding protein are also identical, this difference in structure may account for the observations that 1) chicken gizzard filamin produces parallel actin bundles (27) whereas human ABP-280 induces the formation of branched actin polymers (28), and that 2) chicken gizzard filamin is cleaved by calpain into only two fragments: 240 and 10 kDa (27).
Other reports suggest the existence in a single species of more than one filamin. For example, Gomer and Lazarides (29, 30) described an apparent switch in filamin types during chick skeletal muscle development, and Mangeat and Burridge (31) described the presence of two filamin types in HeLa cells. In addition, two other proteins showing homology with CRF and ABP-280 have been isolated from chicken gizzard, the 450-kDa "fulcin" (32), and the 260-kDa "ABP-260" (33). Partial cDNA sequence is available for each, although in neither case is sequence available for the areas corresponding to the hinge regions.
In this paper, we describe a new 280-kDa protein, -filamin, highly
homologous to ABP-280 and CRF.
-Filamin contains the same
NH2-terminal actin-binding signatures present in the other two proteins as well as in
-actinin (34),
-spectrin (35), and
dystrophin (36). Following the actin-binding domain,
-filamin shares
with ABP-280 and CRF a structure (not present in
-actinin,
-spectrin, and dystrophin) consisting of 24 internally homologous repeats (Figs. 2 and 3). However,
-filamin differs from the other two sequenced filamins in the region of the first of the two hinges; its first hinge sequence is totally distinct from that of ABP-280 (Fig.
4), whereas the reported CRF sequence lacks a first hinge completely.
-Filamin does not contain in its first hinge the calpain cleavage
sequence PQY
TYA present in the first hinge of ABP-280 (6) and is
only 44% identical in the second hinge region, suggesting that the two
proteins may have different calpain cleavage patterns, a possibility
currently under investigation in our laboratory.
Three other human sequences have been reported that are closely related
to ABP-280 and -filamin.
1) In 1993, Leedman et al. (9), using immunoglobulins from
Graves' disease patients, reported the cloning from a human thyroid
library of a cDNA specifying a 195-amino acid protein homologous to
the COOH terminus of ABP-280. This predicted protein has been termed
TABP or thyroid autoantigen. It was suggested that TABP may function in
concert with ABP in the thyroid to link integral membrane
glycoproteins, such as the thyroid-stimulating hormone receptor, to
cytoskeletal actin and thus play a role in signal transduction.
However, TABP protein has not been identified in thyroid tissue, and
further characterization of TABP has not been done. TABP and
-filamin are probably transcribed from the same gene, because the
sequence of TABP and of the corresponding region of
-filamin are
almost identical, and the genetic locus of TABP has been identified as
chromosome 3 (UniGene accession number Hs. 81008).
2) Very recently, Zhang and co-workers (10), using the cytoplasmic loop
region of presenilin-1 as bait in a two-hybrid screen, isolated
cDNA fragments encoding the 358 COOH-terminal amino acids of
ABP-280, as well as an unknown 291-amino acid sequence showing 69%
identity with ABP-280, which they termed "filamin homolog 1" (Fh1).
They mapped the Fh1 gene to chromosome 3. The sequence they reported is
identical to the COOH-terminal 291 amino acids of -filamin except
for two residues (L2312M and G2382E, using the
-filamin residue
numbers), making it very likely that it represents the COOH-terminal
region (most of repeat 22, repeat 23, the second hinge, and repeat 24)
of
-filamin.
3) Maestrini et al. (8) reported two partial cDNA sequences, mapping to chromosome 7, that are homologous to repeats 4-6 and 15-19 of ABP-280. The second of these cDNAs contains the region around the first hinge but lacks any first hinge sequence (Fig. 4). By Northern blotting, an mRNA similar in size to ABP-280 was identified exclusively in skeletal and cardiac muscle. We have confirmed by RT-PCR the existence of such a species and its limited tissue distribution.3 We are currently attempting to isolate and sequence a larger region of this protein, particularly the area that spans the second hinge region, to establish the relationship between this entity and the other filamins.
Filamin-type proteins are not limited to vertebrates. A closely related
actin-binding protein has been identified in Dictyostelium discoideum (37). This 120-kDa protein, referred to as ABP-120, consists of an NH2-terminal actin-binding domain followed
by six filamin-like 100 residue repeats, but lacks any hinge regions, and exists as a 240-kDa homodimer (37). The three-dimensional structure
of repeat 4 has been determined by NMR spectroscopy and consists of
seven -sheets arranged in an immunoglobulin-like fold (38). This is
the only reported structural analysis of a filamin repeat. ABP-120
appears to modulate pseudopod extension in D. discoideum
(39).
These studies and the present report define a group of filamin-related
proteins, which we suggest be termed the filamin superfamily, on the
basis of homology with chicken filamin. Within this superfamily, we
suggest naming well characterized members by species and by order of
discovery and full characterization. Thus, ABP-280 would be termed
human -filamin and our protein would be called human
-filamin.
The term "human
-filamin" should probably be reserved for the
chromosome 7 product.
The filamins appear to function as promoters of actin polymerization
and to connect cell membrane constituents to the actin cytoskeleton.
The former function has been demonstrated for filamins from several
species (40). The latter function has been implied by several
immunohistochemical localization studies in chickens (29, 32) but
investigated in detail only for human ABP-280 (-filamin). The first
membrane protein shown to interact with ABP-280 (
-filamin) was
GpIb
(3, 26, 41). The interaction appears to involve residues
536-568 of the 96-residue cytoplasmic tail of GpIb
(4, 5) and an
area within residues 1850-2136 (from the end of repeat 16 to the
beginning of repeat 20) of ABP-280 (
-filamin) (16). The interaction
of
-filamin with the cytoplasmic tail of GpIb
involves an area
within residues 1862-2148 (from the middle of repeat 17 to the middle
of repeat 20). Therefore, on the basis of the homology between these
two filamins, it is likely that the GpIb
binding sequence is
somewhere between the middle of repeat 17 and the beginning of repeat
20.
In addition to its interaction with GpIb, ABP-280 (
-filamin) has
been shown to associate with other membrane proteins, including the
high affinity IgG receptor Fc
RI (42), the
2-integrin
CD18 subunit (43), presenilin 1 (10), tissue factor (44), and the
calcium-dependent serine endoprotease furin (45), as well as with the cytoplasmic protein SEK-1 (46). Although the binding sites
for Fc
RI and
2-integrin are not known, the regions of ABP-280 (
-filamin) binding to SEK-1 and furin are contained within residues 2282-2454 (repeats 21-23) and residues 1490-1607 (repeats 13-14), respectively. Furthermore, our data and the data of Zhang et al. (10) identify repeats 17-20 and 22-24 of
-filamin as distinct binding regions for the cytoplasmic tails of
GpIb
and presenilin-1, respectively. Thus, despite their strong
internal homology, sufficient unique information resides in different
repeats to create specific binding sites for a number of proteins. We are currently investigating the interactions of
-filamin with other
cellular constituents.
The functions of the filamins are not fully known, but ABP-280 has been
demonstrated to play a crucial role in cellular cytoskeletal and
membrane organization (47), and may also be critical for membrane
expression of some receptors (48). We have published evidence that ECs
synthesize and express on their membranes all four components of the
GpIb/IX/V complex, although the membrane density of these components in
ECs is substantially lower than in platelets (2). -Filamin is
expressed in only trace amounts in platelets, and we observed only
small amounts of GpIb
bound to
-filamin, consistent with the
observations of Ezzell et al. that as much as 90% of
platelet GpIb
is bound to ABP-280 (
-filamin) (26). However,
substantial amounts of both
-filamin and ABP-280 (
-filamin) are
present in ECs, and in ECs we detected relatively large amounts of
-filamin bound to GpIb
. The homology between
-filamin and
ABP-280, as well as our findings and those of Zhang et al.
(10), suggest that these two proteins share a number of functions. On
the other hand, the differences in structure, particularly in the hinge
regions, suggest that
-filamin may have unique effects on
membrane-cytoskeletal interactions, an area under active investigation
in our laboratory.
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ACKNOWLEDGEMENTS |
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Peptides were synthesized at The University of Virginia Biomolecular Research Facility. Nucleic acid sequencing and confocal microscopy were performed at Core Facilities of the Kimmel Cancer Institute, Jefferson Medical College. We are grateful to Dr. Marcia Monteiro and Diana Whitaker-Menezes for their expert technical assistance and to Dr. Kay Heubner (Kimmel Cancer Institute) for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL09163 (to S. S. S.) and fellowships from the Brandywine Valley Hemophilia Foundation and the Delaware Valley Chapter of the National Hemophilia Foundation (to T. T.). This work was presented in part at the 39th Annual Meeting of the American Society of Hematology, Dec. 6-9, 1997, San Diego, CA, and published in abstract form (Takufuta, T., Wu, G., and Shapiro, S. S. (1997) Blood 90, 290a (abstr.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF042166.
¶ To whom correspondence should be addressed: Cardeza Foundation for Hematologic Research, Jefferson Medical College, 1015 Walnut St., Philadelphia, PA 19107. Tel.: 215-955-7786; Fax: 215-955-2366; E-mail: Sandor.Shapiro{at}mail.tju.edu.
1 The abbreviations used are: Gp, glycoprotein; ABP, actin-binding protein; ABPL, ABP-like protein; CRF, chicken retinal filamin; EC, endothelial cell; EST, expressed sequence tag; HUVEC, human umbilical vein endothelial cell; STS, sequence-tagged site; TABP, truncated ABP; vWF, von Willebrand factor; PCR, polymerase chain reaction; RT, reverse transcriptase; PBS, phosphate-buffered saline; kb, kilobase pair(s); bp, base pair(s); nt, nucleotide(s); BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.
2 Information is available on the World Wide Web (http://wwwgenome.wi.mit.edu).
3 T. Takafuta and S. S. Shapiro, unpublished data.
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