(Received for publication, September 9, 1996, and in revised form, November 8, 1996)
From the Joseph J. Jacobs Center for Thrombosis and
Vascular Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, § Children's Hospital Oakland Research Institute, Oakland,
California 94609, ¶ Cardiovascular Research Institute, Department
of Pathology, University of California, San Francisco, San Francisco,
California 94143, and
Experimental Medicine Division, Brigham
and Women's Hospital, Department of Medicine, Harvard Medical School,
Boston, Massachusetts 02115
Actin-binding protein (ABP-280) is a component of
the submembranous cytoskeleton and interacts with the glycoprotein (GP) Ib subunit of the GP Ib-IX complex in platelets. In the
present studies, we have identified the binding site for GP
Ib
in ABP-280. A melanoma cell line lacking ABP-280 was
stably transfected with the cDNAs coding for GP Ib-IX, then
transiently transfected with cDNA coding for various
carboxyl-truncates of ABP-280. Immunocapture assays and
co-immunoprecipitation experiments from detergent-lysed cells showed
that deletion of the carboxyl-terminal repeats 20-24 of ABP-280 had no
effect on GP Ib-IX binding, but deletion of residues 2099 through 2136 within repeat 19 abolished binding. In the yeast two-hybrid system, an
ABP-280 fragment comprising repeats 17-19 bound GP Ib
.
Deletion from either end abolished binding. Individual or multiple
repeats of ABP-280 were expressed as fusion protein in bacteria and
purified; structural folding was evaluated, and binding to GP Ib-IX was
assessed. Binding depended on the presence of repeats 17-19. None of
the individual repeats were able to bind to GP Ib-IX. These findings
demonstrate that residues 1850-2136 comprising repeats 17-19 contain
the binding site for GP Ib-IX.
The activation and association of membrane receptors with the
submembranous cytoskeleton is of key importance in regulating cellular
functions such as adhesion, motility, transmembrane signaling, receptor
distribution, and receptor function. A major component of the
submembranous cytoskeleton is actin-binding protein-280 (ABP-280),1 also known as filamin. In
platelets, ABP-280 cross-links actin filaments and anchors the membrane
skeleton to the plasma membrane by interacting with the cytoplasmic
tail of the von Willebrand factor receptor, the glycoprotein (GP) Ib-IX
complex (1-6). GP Ib-IX is a heterotrimeric complex consisting of two
disulfide-linked subunits, GP Ib
(Mr 140,000) and GP Ib
(Mr 25,000), that are noncovalently associated
with GP IX (Mr 22,000) (7, 8). These
glycoproteins are each transmembrane proteins (9-11). The interaction
of the GP Ib-IX complex with ABP-280 is mediated by a region in the
central portion of the cytoplasmic domain of GP Ib
(5,
6, 12). In other cells of myeloid lineage that do not express GP Ib-IX,
ABP-280 binds the high affinity IgG receptor (Fc
RI) and the
2
integrin (13, 14).
Biochemical and structural analysis of human ABP-280 and cloning of the
human endothelial ABP-280 cDNA reveals a protein of 2647 amino
acids with three functional domains (15). ABP-280 subunits
self-associate head to head (16) using the most carboxyl repetitive
element (15, 17). The opposing amino-terminal end of each subunit
contains an actin-binding domain (15, 17-19). The bulk of ABP-280
forms a semiflexible rod domain and is composed of 24 repeats, each
about 96 residues long, that are predicted to fold into 8 -sheets.
The rod domain of ABP-280 is interrupted twice by short sequence
inserts of 20-40 residues between repeats 15 and 16 and repeats 23 and
24. These regions are postulated to be flexible hinges, and they both
contain a calpain cleavage site. Calpain acts initially at the region
between repeats 15 and 16 to generate an amino-terminal fragment of 190 kDa and a carboxyl-terminal fragment of 100 kDa (20). The 100-kDa
fragment is subsequently cleaved at the site between repeats 23 and 24 to generate 90- and 10-kDa subfragments. Previous reports showed that
in platelet lysates in which ABP-280 has been cleaved, GP Ib-IX
co-immunoprecipitates with the 100-kDa fragment and to a lesser extent
with the 90-kDa fragment (4, 6, 21), suggesting that the binding site
for GP Ib
is located in the carboxyl-terminal portion of
ABP-280.
In the present studies, using three different approaches, we
characterized the region of ABP-280 that interacts with GP
Ib. In the first approach, a melanoma cell line that
lacks ABP-280 (22) was stably transfected with the cDNAs coding for
the three subunits of the GP Ib-IX complex, then transiently
transfected with cDNAs coding for ABP-280 lacking increasing
numbers of amino acids from the carboxyl-terminal end; the ability of
the truncates to associate with GP Ib-IX was determined. An ABP-280
fragment truncated at residue 2136 at the border of repeats 19 and 20 (yielding a fragment lacking 511 carboxyl-terminal residues) retained
GP Ib
binding. In contrast, a fragment truncated at
residue 2099 failed to bind. These results suggest that repeats 20-24 are not required for binding of GP Ib
but that removal
of residues 2099 to 2136 of the carboxyl-terminal third of repeat 19 abolishes binding. As a second approach, the yeast two-hybrid system
was used (23-27). In this system, an ABP-280 fragment of 286 amino
acids, spanning residues 1850-2136 comprising repeats 17-19, bound GP
Ib
. Fragments with deletions at either end of this
fragment were unable to bind. Lastly, repeats 17-19 were expressed in
bacteria individually or as multiple repeats. Repeats expressed
individually or in tandem were unable to bind to GP Ib-IX; as in the
two-hybrid system, a fragment consisting of repeats 17-19 (residues
1850-2136) was required for binding to GP Ib
.
Yeast plasmids containing the GAL4 DNA binding domain
(pGBT9) (26) and the GAL4 activation domain (pGAD424) (26) were obtained from Clontech Laboratories (Palo Alto, CA). The yeast strain
SFY526 (28) (Clontech) was used to assay protein-protein interactions.
All yeast manipulations as well as -galactosidase enzyme activity
assays were performed as recommended by the supplier (Clontech).
For preparation of a GP Ib cDNA-containing plasmid,
a 0.58-kb DNA fragment containing the sequence coding for the
cytoplasmic domain of GP Ib
(nucleotides 1637-2218) (9)
was cloned into pGBT9 (pGBT9-GP Ib
). For preparation of
plasmids containing cDNA encoding fragments of ABP-280, a 3.44-kb
DNA fragment (nucleotides 4927-8313) coding for the carboxyl-terminal
part of ABP-280 (aa 1585-2647) (15) was inserted into pGAD424
(pGAD-ABP3.4). pGAD-ABP3.4 was cut with BglII (all
restrictions enzymes used were purchased from New England Biolabs,
Inc., Beverly, MA), and the resultant 8.7-kb fragment was isolated and
religated. This new plasmid (pGAD-ABP2.1) contained the ABP cDNA
(nucleotides 4927-7017) coding for aa 1585-2282. pGAD-ABP3.4 was also
digested with PstI; the 8-kb fragment was isolated and
religated. The resulting plasmid (pGAD-ABP1.4) contained the ABP
cDNA (nucleotides 4927-6370) coding for aa 1585-2066. Additional
constructs were generated by digesting pGAD-ABP2.1 with
BglII and producing deletions using exonuclease III (U. S. Biochemical Corp.). The fragments were digested, isolated, and ligated
to pGAD424. The clones were sequenced to determine the sizes of the
deletions. Three clones were selected. pGAD-ABP1.8 contained the
cDNA sequence (nucleotides 4927-6753) coding for aa 1585-2194;
pGAD-ABP1.6 (nucleotides 4927-6580) coded for aa 1585-2136; and
pGAD-ABP1.5 (nucleotides 4927-6469) coded for aa 1585-2099. A 0.2-kb
fragment isolated after digesting pGAD-ABP1.6 with PstI was
introduced into pGAD424 to generate pGAD-ABP0.2 (nucleotides 6370-6580
coding for aa 2066-2136). Various fragments of DNA coding for ABP were
generated by PCR, and the resultant PCR products were inserted into
pGAD424 to generate pGAD-ABP0.3 (nucleotides 6232-6580 coding for aa
2020-2136), pGAD-ABP0.5 (nucleotides 6022-6580 coding for aa
1950-2136), and pGAD-ABP0.8 (nucleotides 5722-6580 coding for aa
1850- 2136). The sequences of the amplified fragments were verified by
DNA sequencing analysis.
The
melanoma cells used in these studies were from the human melanoma cell
line M2 lacking ABP-280 (22). The cells were stably transfected with
the cDNAs encoding GP Ib, GP Ib
, and
GP IX, as described previously (29). Subsequently, cells were
transiently transfected by the calcium phosphate method (30) with the
cDNA coding for either full-length or truncated ABP-280. The cells
were harvested 48 h after transfection.
To prepare ABP-280 constructs for transfection into the melanoma cells,
full-length or fragments of cDNA coding for ABP-280 were inserted
into pCDM8 (31). ABP-280 cDNA in the Bluescript SK vector was
digested with XbaI, and carboxyl-terminal truncations were
obtained by incrementally deleting the 3 end with exonuclease III. A
synthetic oligonucleotide, containing stop codons in all three reading
frames, was added. After digestion, the truncated ABP-280 cDNA
fragments were subcloned into pCDM8. The clones were sequenced to
identify the precise base pair at the end of each truncate. The
selected clones were pCDM8-ABP8.03 (nucleotides 1-8030, coding for aa
1-2620); pCDM8-ABP7.9 (nucleotides 1-7916, coding for aa 1-2581);
pCDM8-ABP7.7 (nucleotides 1-7739, coding for aa 1-2523); and
pCDM8-ABP7.3 (nucleotides 1-7340, coding for aa 1-2390). To generate
additional constructs, pGAD-ABP1.6 was digested with SalI
and BsmI, and a fragment of 0.6 kb was isolated. pCDM8-ABP
was digested with BsmI and HindIII, and the 6-kb
fragment was isolated. Both fragments were cloned by a three-way
ligation into pCDM8 cut with HindIII and XhoI.
The resulting construct (pCDM8-ABP6.6) contained the cDNA
(nucleotides 1-6580) coding for aa 1-2136. Another construct was
produced in a similar manner whereby pGAD-ABP1.5 was used as the source
of a 0.5-kb fragment instead of the 0.6-kb of pCDM8-ABP6.6. This
shorter construct (pCDM8-ABP6.5, nucleotides 1-6469) coded for aa
1-2099.
Melanoma cells were harvested by treatment
with 1 mM EDTA (Sigma) and stained with 2 µg/ml of monoclonal antibody against GP Ib (MAb Ib-23)
(generously provided by Dr. B. Steiner, Hoffmann-La Roche) (32) and
with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (5 µg/ml) (Vector Laboratories, Inc., Burlingame, CA). The samples were
analyzed by a FACScan flow cytometer (Becton Dickinson, San Jose,
CA).
To
determine whether ABP-280 fragments expressed in melanoma cells
associated with GP Ib-IX, cells were harvested with EDTA, and lysed in
a Triton X-100-containing buffer; the detergent-soluble fractions were
isolated by centrifugation as described previously (29). Monoclonal
antibody ABP-4 (MAb ABP-4) (4, 15) or mouse IgG (Sigma Immuno Chemical
Co.) were covalently coupled to 10 µM polyacrylamide
immunobeads (Bio-Rad) according to the manufacturer's instructions.
After preclearing with mouse IgG-coupled immunobeads, the Triton
X-100-soluble fractions were incubated with MAb ABP-4-coated beads.
After extensive washing, the immunoprecipitated proteins were removed
from the beads and analyzed on Western blots (29). The blots were first
incubated with a monoclonal antibody against GP Ib (MAb
Ib-4, courtesy of Dr. B. Steiner) (32) and then reprobed with a
monoclonal antibody against ABP-280 (MAb ABP-4) (4, 15).
Antigen-antibody complexes were detected by enhanced chemiluminescence
using Amersham's ECL system.
To determine whether ABP-280 fragments expressed in bacteria associated with GP Ib-IX, ABP-deficient melanoma cells, expressing GP Ib-IX, were lysed as described above. Triton X-100-soluble fractions were incubated for 5 h with GST fusion protein-agarose bead complexes (Sigma).
Immunocapture AssayThe wells of microtiter plates were
coated with 100 µl of MAb ABP-4 (2 µg/ml) overnight at 4 °C. The
plates were saturated for 4 h at 4 °C with 3.5% bovine serum
albumin. Varying concentrations of Triton X-100-soluble fractions from
melanoma cells were added to the wells and incubated overnight at room
temperature. GP Ib associated with ABP-280 was detected
by sequential incubation with a rabbit anti-GP Ib-IX antibody (1:1000,
1 h) (courtesy of Dr. B. Steiner) (32), biotinylated anti-rabbit
IgG (Amersham) (1:2000, 1 h), streptavidin-alkaline phosphatase
(Pierce, Rockford, IL) (1:10000, 1 h), and the substrate for
alkaline phosphatase, p-nitrophenyl phosphate (Pierce). The
color development was measured in a spectrophotometer at 410 nm
(Dynatech Laboratories Inc., Chantilly, VA).
ABP-280 cDNAs corresponding to the desired amino
acids were generated by PCR and ligated into a pGEX vector (Pharmacia
Biotech Inc.). All 3 primers contained stop codons in all three
reading frames to insure proper termination of translation. The
sequences of the amplified fragments were verified by DNA sequencing
analysis.
Fusion proteins were expressed in Escherichia coli.
Translation of the vector was induced with 1 mM isopropyl
-thio-galactopyranoside for 3 h. Cells were harvested by
centrifugation at 6600 × g for 10 min and lysed by
sonication; the resulting supernatant was passed over a
glutathione-agarose column. Before evaluation of the secondary
structure, GST was cleaved by thrombin (Sigma) (25 units/liter of bacterial culture). Released proteins were collected by
centrifugation at 2500 × g for 5 min, and purity was
evaluated by SDS-polyacrylamide gel electrophoresis. Protein
concentrations were determined using the BCA assay (Pierce).
Polypeptides expressed in bacteria at final concentrations of 1 mg/ml were extensively dialyzed in 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.5, and brought to room temperature before spectral analysis. Measurements were performed in an Aviv 60DS spectropolarimeter at room temperature. Cuvette path lengths of both 1 and 0.1 cm were used, and measurements were taken at step sizes of 0.5 nm. Secondary structure was determined using the software program Theta, which correlates spectra and predicted secondary structure.
As a first approach to
identifying the region of ABP-280 that interacts with GP
Ib, we used the melanoma cell line M2 deficient in
ABP-280 (22). M2 cells were stably transfected with the cDNAs
encoding GP Ib
, GP Ib
, and GP IX. Even in
the absence of ABP-280, the three subunits were expressed, formed a
complex, and were inserted into the plasma membrane. Fig.
1 demonstrates the surface expression of GP Ib-IX in
cells stained with an antibody against GP Ib
.
Similar results were obtained with an antibody against GP IX (data not
shown).
The GP Ib-IX-expressing cells were then transiently transfected with
the constructs shown in Fig. 2 containing cDNAs
coding for ABP-280 lacking increasing numbers of amino acids from the carboxyl-terminal end. The amount of ABP-280 carboxyl truncates expressed in the cells transfected with the different constructs was
very similar (data not shown). To determine whether GP Ib-IX associated
with the various truncated forms of ABP-280, the transfected cells were
lysed in a Triton X-100-containing buffer. The ABP-280 fragments were
captured with an anti-ABP antibody absorbed onto microtiter plates, and
the amount of GP Ib-IX associated with the fragments was determined.
Fig. 3A shows that like full-length ABP-280
in stably transfected cells (construct 1), ABP-280 truncates expressed
in cells transiently transfected with constructs 2-5 (lacking 27 to
257 carboxyl-amino acids contained within the last third of repeat 22 through repeat 24) were recovered in association with GP Ib-IX.
Similarly, a protein lacking repeats 20-24 (deletion of 511 carboxyl-terminal amino acids), expressed in cells transiently transfected with construct 6, was able to bind to GP Ib-IX just as well
as full-length ABP-280 (Fig. 3B). In contrast, an ABP-280 fragment missing 548 amino acids (lacking repeats 20-24 plus the carboxyl-terminal third of repeat 19), expressed just as efficiently as
the fragment lacking 511 carboxyl-terminal amino acids (Fig. 3,
inset, compare lanes 7 and 6), did not
bind to GP Ib-IX (Fig. 3B, column 7). Similar results were
obtained when GP Ib was captured with an anti-GP
Ib
antibody and the amount of ABP-280 associated was
measured (data not shown).
The ability of the ABP-280 fragments to interact with GP Ib-IX in
melanoma cells was also assessed by immunoprecipitating ABP-280 from
detergent-lysed cells. In this experiment, GP Ib-IX-expressing cells
were transiently transfected with the cDNA encoding full-length or
truncated ABP-280. Full-length ABP-280 (Fig. 4B,
lane 2), as well as proteins missing 511 and 548 amino acids from
their carboxyl termini (Fig. 4B, lanes 3 and 4),
were immunoprecipitated using an anti-ABP antibody. GP
Ib co-immunoprecipitated with full-length ABP-280 and
with ABP-280 lacking 511 carboxyl-amino acids (Fig. 4A, lanes
2 and 3). However, GP Ib
did not
co-immunoprecipitate with ABP-280 lacking 548 carboxyl-amino acids
(Fig. 4A, lane 4).
These results suggest that repeats 20-24 of ABP-280 are not required for binding of ABP-280 to GP Ib-IX, but that residues 2099-2136 in the carboxyl-third of repeat 19 are essential.
Further Characterization of the GP IbAs a second approach to
identifying the GP Ib-IX binding site on ABP-280, we used the
two-hybrid system (23-27). Based on previous findings, suggesting that
the binding site for GP Ib was located in the
carboxyl-terminal third of ABP-280 (4, 21), the cDNA encoding the
carboxyl-third of ABP-280 (aa 1585-2647, consisting of the middle of
repeat 14 through repeat 24) was fused to the GAL4 activation domain.
The cDNA encoding the cytoplasmic domain of GP Ib
was fused to the GAL4 DNA binding domain. The recombinant plasmids were
co-transformed into the SFY526 yeast strain. The interaction between GP
Ib
and ABP-280 reconstituted the function of GAL4;
-galactosidase activity was induced, resulting in the generation of
blue colonies (Fig. 5). Neither fusion protein alone was
able to activate the transcription of the reporter gene. These results
confirmed that the binding site for GP Ib
was contained
within the carboxyl-terminal third of the molecule. As in the melanoma
cells, deletion of the last 511 carboxyl-terminal amino acids of the
ABP-280 subunit (aa 1585-2136, consisting of the middle of repeat 14 through repeat 19) did not affect its binding to GP Ib
in the two-hybrid system (Fig. 5). Binding was lost, however, when just
37 more residues were deleted (deletion of the last 548 carboxyl-terminal amino acids).
The results presented thus far demonstrate that a fragment of ABP-280
consisting of the middle of repeat 14 through repeat 19 (residues
1585-2136) can bind GP Ib. To determine which part of
the amino-terminal portion is required for binding, constructs containing cDNA coding for ABP-280 fragments with increasing
deletions from the amino-terminal end were prepared. A truncated
fragment of 286 amino acids containing residues 1850-2136 (comprising
repeats 17-19) was still able to bind to GP Ib
(Fig.
5). However, further deletions of this fragment from its amino-end
abolished the binding to GP Ib
(Fig. 5); a truncate
consisting of residues 1950-2136 within repeats 18-19 failed to bind
to GP Ib
. When a truncate consisting of residues
1850-2136 of ABP-280 was expressed, blue colonies could be detected
after 30 min, whereas a 15-18-h incubation was necessary to detect an
interaction with a truncate composed of residues 1585-2136. With a
liquid assay, 166
-galactosidase units were generated with the
shorter construct compared to 3.4 with the longer construct (Fig.
5).
It is conceivable that the shorter fragments of
ABP-280 expressed in yeast (aa 1950-2136 and aa 2020-2136) may have
lost their ability to bind to GP Ib-IX because of loss of their
secondary structure. Thus, an additional assay using GST-fusion
proteins was used. Individual repeats 18 (aa 1950-2038), 19 (aa
2039-2135), and 19 + 32 aa (aa 2039-2167), or multiple repetitive
elements 18-19 (aa 1950-2135) were expressed as GST-fusion proteins
in bacteria and purified; their structural folding was evaluated by
circular dichroism. Human ABP-280, a protein composed primarily of
-structure, has a single minimum wavelength of 218 nm (33). In the
present study, evaluation by the Theta algorithmic program indicated
that purified human ABP-280 had approximately 13%
-helix, 62%
-sheet, and 25% random coil. Purified polypeptides also had a
minimum at 218 nm, and the percentage of
-helix was between 11 and
12%,
-sheet was between 59 and 62%, and random coil was between 28 and 30%, suggesting that the purified recombinant repeats achieved
secondary structure comparable to that of purified human ABP-280.
Purified GST fusion proteins coupled to agarose beads were incubated
with extracts from Triton X-100-lysed, ABP-deficient melanoma cells
expressing GP Ib-IX. GST beads alone (Fig. 6, lane 2) did not bind GP Ib; neither did repeat 18 alone
(Fig. 6, lane 3), repeat 19 alone (Fig. 6, lane
4), the tandem repeats 18-19 (Fig. 6, lane 5), or repeat 19 + 32 aa (Fig. 6, lane 6). In contrast, repeats 17-19 expressed
as GST-fusion protein (Fig. 6, lane 7) bound GP
Ib
. These results confirm the results obtained with the
two-hybrid system that the binding site for GP Ib
on
ABP-280 is located in repeats 17-19 between residues 1850 and
2136.
The goal of the present study was to identify the domain on
ABP-280 that binds GP Ib. Three different approaches
have lead to the same conclusion that the binding site for GP
Ib
is located within repeats 17-19 of ABP-280. In the
first approach, melanoma cells deficient in ABP-280 but expressing GP
Ib-IX were transfected with various cDNAs directing the synthesis
of carboxyl-deleted ABP-280. ABP-280 truncates lacking
carboxyl-terminal residues from 27 to 511 associated with GP Ib-IX in
these cells, but further deletion of 37 residues abrogated GP Ib-IX
binding. The deletion of the carboxyl-third of the repeat 19 (residues
2099-2136), therefore, disrupts GP Ib
binding to
ABP-280. A second approach used the yeast two-hybrid system
(23-27). Portions of the carboxyl terminus of ABP-280 were
co-expressed along with the cytoplasmic tail of GP Ib
.
The smallest fragment of ABP-280 that bound GP Ib
was
composed of repeats 17-19 (residues 1850-2136). Further deletions from either end of this fragment abolished the binding to GP
Ib
. These results demonstrate that the GP
Ib
binding site is near the middle of the 90 kDa rod
fragment. These results were confirmed by expressing individual or
multiple ABP-280 repeats as fusion protein in bacteria and testing
their binding to GP Ib-IX. Repeats 17-19 (residues 1850-2136), but
not repeat 18 or 19 or the tandem 18-19, bound to GP Ib-IX. Circular
dichroism measurements indicated that the folding of the expressed
proteins was comparable to that in native ABP-280. The combination of
these three approaches strongly suggests that the binding site on
ABP-280 for GP Ib
is contained in repeats 17-19
(residues 1850-2136). This is somewhat surprising because a great
degree of conformity to the consensus repeat exists in repeats 17 and
19, and we would expect the binding site to be located in a less
conserved region. A bigger divergence exists in repeat 18, where the
regular pattern is interrupted by a 9-residue deletion at the initial
portion of the repeat. This divergence from the consensus sequence may play a role in the binding of GP Ib
.
The rod domain of ABP-280 is interrupted at two points by sequence
inserts that contain a calpain cleavage site. Earlier studies showed
that in platelet lysates in which ABP-280 was cleaved by calpain, GP
Ib-IX remained associated with the 100-kDa carboxyl portion of the
polypeptide chain and to a lesser extent with its 90-kDa
calpain-generated subfragment (4, 6, 21). This suggested that GP Ib-IX
might bind near the calpain cleavage site in the hinge just before
repeat 24 (15). Alternatively, calpain cleavage could perturb the
conformation of the 90-kDa subfragment and thereby diminish GP Ib-IX
binding. The location of the GP Ib binding site
determined in the present study suggests the latter mechanism to
explain the decreased amount of GP Ib
associated with
the 90-kDa subfragment.
ABP-280 has proved difficult to dissociate from the GP Ib-IX
complex in detergent-lysed platelets (1-4, 34). This observation stands in marked contrast to the 1 × 107
M
1 affinity of ABP-280 for GP Ib-IX measured
in vitro (5). An experimental observation may shed some
light on this discrepancy. The interaction between the fragment of
ABP-280 formed by repeats 17-19 (residues 1850-2136) and GP
Ib
was relatively strong in the two-hybrid system
compared to the one obtained with larger fragments from ABP-280 (Fig.
5). This might suggest that binding to GP Ib
can be
modulated by flanking sequence on both ends of repeats 17-19. Many
other factors could, of course, contribute to the weaker signal
obtained in the two-hybrid system with larger fragments; for example,
these fragments may be folded inappropriately, they may be less stable
than the shorter ones, they may be expressed less efficiently than the
smaller fragments, or there may be differences in the accessibility of
the interacting domains to each other or accessibility of the
activation domain to the transcriptional machinery.
Human GP Ib-IX has been expressed in hamster and mouse nonhematopoietic
cells in a form that associated with ABP-280 (35). Thus, the domain on
ABP-280 that associates with the cytoplasmic domain of GP
Ib appears to have been conserved. ABP-280 is a
prominent component of many different cell types (36, 37), and it now
appears that the rod of this protein may act as a scaffolding to
collect and tether diverse molecules at the cytoplasmic surface of the
plasma membrane. Membrane association is conferred in platelets and
megakaryocytes by the binding of repeats 17-19 to GP Ib-IX complexes.
Other membrane receptors that bind to ABP-280 are the IgG Fc receptor I
(Fc
RI) (13) and integrin
2 (14). Sequence comparison
between the cytoplasmic tails of GP Ib
, Fc
RI, and
integrin
2 has, however, revealed no sequence
conservation (9, 14, 38). Thus, the domain on ABP-280 implicated in the
interaction with Fc
RI and integrin
2 is likely to be
different from the one interacting with GP Ib
. Other
proteins now known to bind along ABP-280 include small GRP-binding
proteins, the endosomal endoproteinase
furin,2 and the transcription factor
ATFa.3 Future studies will be needed to
characterize the way in which ABP-280 interacts with additional
membrane glycoproteins and to elucidate the importance of ABP-280 in
regulating the properties of these plasma membrane receptors.
The authors are grateful to Dr. J. López for the constructs coding for GP Ib-IX, Dr. B. Steiner for antibodies, and Gene Lazuta for editorial assistance.