From the Departments of Biochemistry and Molecular Biophysics, § Infectious Diseases, and ¶ Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Integrin-associated protein (IAP; CD47) is a
thrombospondin receptor that forms a signaling complex with
The thrombospondins
(TSPs)1 are a family of
multidomain, secreted glycoproteins whose production is differentially
regulated during development, wound repair, inflammation,
tumorigenesis, and other states in which rapid changes occur in cell
proliferation and migration (1, 2). Many of the apparently diverse
effects of TSP proteins can be ascribed to the presence of different
complements of receptors for the several domains of TSP on different
cell types. Integrin-associated protein (IAP or CD47) is a receptor for
the carboxyl-terminal cell binding domain of TSP1, which contains the
active CD47 agonist peptide RFYVVMWK (3, 4). This sequence is well
conserved among species and TSP isoforms (2), suggesting that all TSP
family members use CD47 as a receptor. CD47 is an unusual member of the
IgG superfamily of receptors. It consists of a single IgGv
extracellular domain and five transmembrane segments terminating in a
short, alternatively spliced cytoplasmic tail (5). Monoclonal
antibodies (mAbs) against CD47 block signaling events such as the
increase in intracellular Ca2+ that occurs when endothelial
cells spread on matrix proteins containing RGD sequences (6). Further,
anti-CD47 mAbs can block the transendothelial (7) and transepithelial
(8) migration of polymorphonuclear nucleophiles, and CD47 knockout mice
have a severe host defense defect (9). CD47 appears to be involved in
processes related to integrin-mediated cell spreading (4, 10) and
motility (3, 11) in a number of cell types.
In several biological settings, the binding to CD47 of TSP1, its cell
binding domain, or the agonist peptide 4N1K (KRFYVVMWKK) can stimulate
the "activation" of integrins to a higher affinity/avidity state.
For example, the 4N1K peptide stimulates platelet spreading on
fibrinogen-coated surfaces and induces aggregation of platelets via
activation of the integrin The current study was undertaken to determine if the integrin-CD47
complex might functionally associate with a pertussis toxin-sensitive G
protein. The data presented here indicate that not only does such an
association exist, it has functional consequences for the regulation of
GTP binding and cyclic AMP levels. Furthermore, a negative heterotropic
effect of GTP Materials--
The human melanoma cell line C32 (ATCC CRL 1585)
was cultured as described (4), and all reagents were as described by
Gao et al. (4). Pertussis toxin (List Biologicals) treatment
of C32 cells was carried out overnight at 60 and 300 ng/ml media. OV10
cells and stable transfectants derived from them have been described
(3, 9). Antibodies against G proteins have the following specificities:
856, Gi Immunoprecipitations--
Crude membrane fractions (100,000 × g pellets) were solubilized with the indicated detergents
at 4 °C, and the soluble fraction was incubated with the indicated
primary antibody for 1-3 h on ice. Appropriate secondary antibodies
coupled to agarose were used to collect the bound antigens, and the
complexes were washed extensively, solubilized in boiling SDS sample
buffer with reduction, and subjected to SDS-PAGE and blotting onto
nitrocellulose. Blots were probed with the indicated antibodies as
described (4). Affinity labeling in the presence of the indicated
compounds with iodinated 4N1K peptide was as described (3, 4).
Affinity Chromatography--
His6-4N1K peptide (1 mg) was adsorbed onto fresh nickel-NTA matrix (200 µl) and incubated
2 h with a detergent lysate of C32 cell membranes at 4 °C.
After washing with 30 ml of HEPES-buffered saline buffer containing the
same detergent, stepwise elution with the indicated concentrations of
imidazole was initiated (see Ref. 10 for details). After SDS-PAGE,
blots were probed with anti-G GTP CD47 and Gi Proteins Co-immunoprecipitate--
To
investigate the possible association of CD47 and its integrin partner
with G proteins, octyl glucoside extracts of C32 cell membranes were
affinity-labeled with 125I-4N1K peptide (3, 11), resulting
in specific labeling of the 52-kDa CD47 (Fig.
1). This labeled lysate was then
immunoprecipitated with antibody that recognizes G Association of Gi and CD47 Is Pertussis
Toxin-sensitive--
Pertussis toxin catalyzes the ADP-ribosylation of
Gi Gi Copurifies with the CD47-Integrin Complex--
In
an independent approach, we used affinity chromatography to isolate
CD47-containing detergent-soluble complexes. 4N1K and 4NGG peptides
were synthesized with amino-terminal hexahistidine tags and bound to
nickel-NTA beads. Detergent lysates of C32 membranes were adsorbed with
these affinity beads, washed, and then eluted stepwise with increasing
concentrations of imidazole. As seen in Fig.
3A, Gi The Gi Activation Affects CD47 Affinity for
4N1K--
To investigate more quantitatively this negative
heterotropic effect of GTP on the 4N1K-CD47 interaction, the amount of
CD47 bound to the 4N1K charged nickel-NTA beads was quantified using an
125I-labeled mAb, 2D3, which binds CD47 but does not
perturb either ligand binding or signaling from CD47 (4, 10). Detergent lysates of C32 membranes were incubated with the 4N1K-charged nickel-NTA agarose beads along with nucleotides or AlF4;
4NGG-charged beads served as a control. After incubation and rapid
washing of the beads, the amount of CD47 bound was determined with
125I-2D3 mAb. As seen in Fig.
4A, GTP 4N1K Decreases Intraplatelet Cyclic AMP via Gi--
We
have shown that 4N1K activates platelets via a signaling pathway, which
includes pertussis toxin-sensitive activation of SYK (10). Elevated
levels of intraplatelet cyclic AMP inhibit activation, and a key
feature of activation is a rapid decrease in cAMP levels. Thus,
platelets maintained in prostaglandin E1, whose receptor is
coupled to Gs, are prevented from activating (19). We
tested the effect of CD47 activation on cyclic AMP levels in suspended,
unstirred platelets, thus preventing their aggregation and the
subsequent wave of integrin-dependent outside-in signaling
(19). As seen in Fig. 5, the
prostaglandin E1-treated platelets have a high level of
intracellular cyclic AMP (CTL), and the control peptide 4NGG
has no effect. In contrast, 4N1K (50 µM) treatment for 1 or 15 min dramatically lowers intraplatelet cyclic AMP levels to nearly
the same extent as 5 µM thrombin receptor peptide, a
strong activator of platelets. Upon treatment of the platelets with 100 ng/ml of pertussis toxin for 2 h prior to stimulation, the effect of 4N1K on cyclic AMP levels is obliterated. Thus, not only
does CD47 associate with Gi, but it also acts via
Gi to rapidly decrease intraplatelet cyclic AMP even in the
face of the elevated levels stimulated by prostaglandin
E1.
CD47 Ligation Stimulates GTP
To confirm that the stimulation of GTP binding by 4N1K is in fact due
to binding of the peptide to CD47 and not to a direct action on the G
protein or another non-receptor-mediated effect of the peptide, we used
fibroblasts isolated from CD47-deficient mice (9) as a control. As seen
in Fig. 6C, 4N1K stimulates GTP binding to membranes
prepared from CD47-deficient fibroblasts that had been transfected with
a human CD47 expression construct but not to the untransfected
CD47-deficient membranes. Lysophosphatidic acid, which binds to a G
protein-coupled receptor on fibroblasts was tested as a positive
control and stimulated GTP The coupling of CD47 to Gi protein activation is
consistent with our knowledge of the biological actions of TSP and
CD47. CD47 functions as a chemotaxis receptor (3) for TSP and 4N1K, and
many receptors for chemotactic ligands are 7TMS receptors that are
coupled to G (usually Gi) proteins (18). CD47 on platelets is a costimulatory receptor for Other signaling events thought to emanate from CD47 include
Ca2+ fluxes (6) and activation of protein kinase C and
phosphatidylinositol 3-kinase (4). All of these events are regulated by
7TMS receptors coupled to heterotrimeric G proteins (21).
Interestingly, all of these activities have also been implicated in
integrin regulation in a variety of systems (22). Our recent data show
that CD47 can associate with and/or modulate integrins of the
Aside from the physical association of integrins, CD47, and
Gi proteins, other factors support the concept of a
functional association in which CD47 communicates directly with
Gi. First, treatment of platelets (Fig. 5) with 4N1K
initiates a precipitous drop in intraplatelet cyclic AMP levels. This
signaling event is common to all of the agonists that activate
platelets through G protein-coupled receptors (19). We have also
observed a similar fall in intracellular cyclic AMP in other cell types
that respond to TSP-1 stimulation with integrin
activation.2 Second, the agonist peptide 4N1K rapidly
stimulates GTP Based on the data presented here, we propose a model for CD47 action in
which the integrin-CD47 heterotrimer functions as an ad hoc
serpentine or 7TMS receptor, each of the integrin subunits contributing
one transmembrane segment and CD47 contributing its five. This
heterotrimeric receptor would then activate heterotrimeric G
proteins in much the same way as classical 7TMS receptors. This hypothesis garners support from a number of additional observations as
follows. (i) The stability of the integrin-CD47 complex is consistent
with the formation of a seven-helix bundle within the core of the
membrane. The integrin-CD47 complex survives solubilization with
several different detergents (including radioimmune precipitation buffer under some conditions) and the extensive washing associated with
affinity chromatography. SDS gels of material eluted from the 4N1K
columns reveal the integrin chains and CD47 as the major protein-stained bands.4 (ii)
Methods that allow detection of CD47-G protein complexes from C32 and
platelet membranes detect little or no CD47-G protein complex when
applied to red blood cell
membranes.5 Red cells contain
both CD47 and Gi proteins but no integrins (25), suggesting
that the integrin may be necessary to stabilize the CD47-G protein
complex. However, there may be integrin-independent actions of CD47 in
other cells such as T cells (26). (iii) The bundle of seven
transmembrane segments need not be covalently connected for even
classical G protein-coupled receptors to bind ligand and activate G
proteins. Proteolysis of the In summary, we report that the CD47-integrin complex includes one or
more heterotrimeric Gi proteins. We provide functional evidence that there is a direct interaction between CD47 and the G
protein as evidenced by the following: (i) a negative heterotropic effect of GTP and AlF4 on the binding of 4N1K by CD47; (ii)
the fact that 4N1K treatment of cells causes a rapid, pertussis
toxin-sensitive drop in intracellular cyclic AMP; and finally (iii)
4N1K specifically stimulates the binding of a radiolabeled GTP analog
to membranes in a CD47-dependent fashion. Given the
ubiquitous distribution of integrins, CD47, and TSP family members,
these results provide a far reaching paradigm for many of the
biological actions of TSP proteins and suggest a novel route for G
protein activation leading to the modulation of integrin function in
many systems.
3 integrins resulting in enhanced
v
3-dependent cell spreading
and chemotaxis and, in platelets,
IIb
3-dependent spreading and
aggregation. These actions of CD47 are all specifically abrogated by
pertussis toxin treatment of cells. Here we report that CD47, its
3 integrin partner, and Gi proteins form a
stable, detergent-soluble complex that can be recovered by
immunoprecipitation and affinity chromatography. Gi
is
released from this complex by treatment with GTP or AlF4.
GTP and AlF4 also reduce the binding of CD47 to its agonist peptide (4N1K) derived from thrombospondin, indicating a direct association of CD47 with Gi. 4N1K peptide causes a rapid
decrease in intraplatelet cyclic AMP levels, a
Gi-dependent event necessary for aggregation.
Finally, 4N1K stimulates the binding of GTP
35S to
membranes from cells expressing IAP and
v
3. This functional coupling of CD47 to
heterotrimeric G proteins provides a mechanistic explanation for the
biological effects of CD47 in a wide variety of systems.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIb
3 as judged
by the enhanced binding of the ligand mimetic mAb PAC-1 (10). When C32
human melanoma cells attach to a sparsely coated vitronectin
substratum, TSP1 and 4N1K dramatically stimulate the rate of
v
3-dependent cell spreading
(4). Further, 4N1K peptide is a chemoattractant of endothelial cells
migrating on a gelatin/RGD-containing matrix to which they attach via
v
3 (3). The chemotaxis of vascular smooth
muscle cells toward soluble collagen is also stimulated by 4N1K. In
this case,
2
1, a collagen receptor, is
modulated by CD47 (11). In each of these systems, the augmentation of integrin function is selectively blocked by pretreatment of the cells
with pertussis toxin, which disables signaling through heterotrimeric G
proteins of the Gi family.
S and AlF4 on ligand binding to CD47
suggests that the association of the integrin-CD47 complex with
Gi is direct.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,2,3; BO87, Gi
1 and Gi
2; B600, G
1 and G
2 (12).
Peptides were synthesized, purified, and verified by mass spectrometry
by the Washington University Protein and Nucleic Acid Chemistry
Laboratory. Platelets were obtained from the St. Louis Red Cross or by
venipuncture of healthy volunteer donors under institutional
guidelines. They were prepared as described by Chung et al.
(10).
and anti-CD47 antibodies.
To determine the amount of CD47 bound to the 4N1K affinity matrix in
the presence of various nucleotides, the Triton X-100 (1% w/v)
detergent-soluble fraction of C32 membranes was incubated with
His6-4N1K-charged nickel-NTA beads (or
His6-4NGG control beads) for 2 h in the presence of the indicated nucleotides or AlF4, all at 60 µM. The beads were rapidly washed and incubated with
125I-mAb 2D3 against CD47 for 1 h. After washing, the
beads were counted in a Beckman
-counter. Cyclic AMP in platelets
was quantified after ethanol extraction with an enzyme-linked
immunosorbent assay kit from Amersham Pharmacia Biotech following the
manufacturer's protocol.
35S Binding Assay--
K562 cells expressing
v
3 integrin (13) or murine fibroblasts
(9) were disrupted by nitrogen cavitation, and membranes were prepared
according to Ref. 14. The lysis buffer contained 20 mM
HEPES, pH 8.0, 1 mM EDTA, 2 mM
MgCl2, and a protease inhibitor mixture (4). Similar
results were obtained when cells were lysed by freeze-thaw cycles.
GTP
35S (NEG030H; NEN Life Science Products) binding was
determined in a filtration assay essentially as described by Sternweis
et al. (15) in a reaction mixture containing the following:
50 mM HEPES, pH 8.0, 100 mM NaCl, 0.75 mM MgCl2, 0.1 mM EDTA. Carrier-free GTP
35S was present at 3-5 nM final
concentration. Binding of GTP
35S occurred at 30 °C;
reactions, which contained equal amounts of membrane protein (~5-10
µg), were stopped; and membranes were filtered and washed with
ice-cold reaction buffer (15). Membrane protein was determined with a
Bradford Assay kit from Bio-Rad.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits (12). As seen in Fig. 1, the labeled 52-kDa CD47 was recovered
in the precipitate (lanes 1 and 6) but
not when nonimmune rabbit IgG was used (lane 2).
The labeling of the precipitated protein was prevented by excess cold
4N1K peptide during the affinity labeling reaction (lane
3), and no labeled protein was recovered when the
125I control peptide 4NGG (KRFYGGMWKK) was used for
labeling instead of 4N1K (lane 4). In addition,
recovery of labeled CD47 was greatly reduced by preincubation of the
anti-G
antibody with the antigenic peptide
(lane 5). In the converse experiment, detergent lysates of C32 cells and human platelets were immunoprecipitated with
two different anti-CD47 mAbs (B6H12 and 1F7), and the precipitates were
analyzed for the presence of G proteins by Western blotting of SDS gels
(Fig. 2A). Both anti-CD47 mAbs
coprecipitated 40-kDa G
and 36-kDa G
subunits from both cell types. As a further control, OV10 ovarian
carcinoma cells, which express no CD47 (3), were used. When these cells
are transfected with
3 integrin cDNA, resulting in
expression of
v
3, no G
protein is recovered in anti-CD47 (1F7) immunoprecipitates (Fig.
2B, lane 3). However, when CD47
cDNA is expressed in these cells, G
is easily
detected in the CD47 complex (lane 1).
Coexpression of CD47 along with
v
3
cDNA leads to an increase in the amount of G
recovered in the anti-CD47 precipitate (lane 2).
The amount of G
expression was comparable in these cell
lines (not shown). The magnitude of the increase varies somewhat from
one experiment to another ranging from the ~2-fold increase shown in
Fig. 2B to nearly 10-fold in some experiments. Thus, a
relatively stable complex containing CD47 and heterotrimeric G proteins
exists in octyl glucoside extracts of C32 and platelet membranes. The
G
subunits, which complex with CD47 comigrate with
purified recombinant Gi standards (Fig. 2) and also react
with an antibody (B087; Ref. 12) specific for Gi
-1 and
-2 (not shown). This is consistent with the pertussis toxin inhibition
of CD47-dependent signaling responses (4, 10) and earlier
reports that chemotactic responses of cells to TSP1 are pertussis
toxin-sensitive (16).
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Fig. 1.
Immunoprecipitation of affinity-labeled CD47
with anti-G antibody. A crude
membrane fraction of C32 cells was treated with 30 mM
octyl-
-D-glucopyranoside, and the soluble fraction was
affinity-labeled with 125I-4N1K peptide (lanes
1-3 and 5-7) or 125I-4NGG peptide
(lane 4) and then immunoprecipitated
(IP) with a polyclonal antibody (856) against
G
peptide GAGESGKSTIVKQMK (lanes 1 and 3-6) or rabbit IgG (lane 2).
Labeled CD47 in the precipitates was detected by SDS-PAGE and
autoradiography. For lane 3, the affinity
labeling was performed in the presence of excess unlabeled 4N1K
peptide. In lane 5, the anti-G
antibody was preincubated with the antigenic peptide. Lane
7 is the labeled membrane lysate before
immunoprecipitation.
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Fig. 2.
Immunoprecipitation of
G and
G
with anti-CD47 antibodies.
A, the octyl-
-D-glucopyranoside (30 mM) soluble fractions of C32 and platelet membranes were
immunoprecipitated (IP) with mAb B6H12 or 1F7 against CD47
or mouse IgG control antibody. After SDS-PAGE, blots were probed with
antibodies against G
(856, top
row) or G
(B600, bottom
row) (12). Standard recombinant G protein subunits (prepared
as in Ref. 16) are shown in the left lane.
B, human ovarian carcinoma cells (clone OV10), which do not
express CD47, were transfected with cDNA encoding the
3 integrin subunit (lanes 3 and
6), CD47 alone (lanes 1 and
4), or CD47 plus the
3 subunit
(lanes 2 and 5) as indicated (13).
Detergent-solubilized membranes from these cells were
immunoprecipitated with either anti-CD47 mAb 1F7 or control mouse IgG.
Recombinant Gi
subunit (lane 7)
was run on the gel as an internal standard. Expression of
3 in these cells allows functional reconstitution of
v
3. The levels of endogenous
Gi
were the same in the three OV10-derived cell lines.
C, C32 cells were treated in culture overnight with either
60 or 300 ng/ml Bordetella pertussis toxin (PTX)
or its inactive B oligomer (B-OLI). Membranes were prepared,
solubilized, and immunoprecipitated with anti-CD47 mAb 1F7 as above.
The blots were probed with the anti-G
or
-G
antibodies.
subunits and prevents productive interaction of
Gi with receptor (17). C32 cells were treated overnight
with either 60 or 300 ng/ml pertussis toxin or its inactive, but
surface binding, B oligomer. Membranes were prepared from these cells,
and detergent lysates were immunoprecipitated with anti-CD47 mAb 1F7.
As seen in Fig. 2C, both concentrations of active toxin
cause a decrease in the amount of G protein
- and
-subunits
recovered in the CD47 complex. Interestingly, the small amount of
G
remaining in the complex after pertussis toxin
treatment migrates at a slightly higher Mr on
SDS gels (18), suggesting that ADP-ribosylated G
retains
a marginal affinity for the CD47 complex. Thus, like 7TMS receptors
that activate Gi proteins, the signaling from CD47 as well
as the association of the G protein is sensitive to pertussis
toxin-mediated ADP-ribosylation.
subunits
(solubilized in two different detergents) eluted from a 4N1K column
along with CD47 at very high imidazole concentrations (0.5-1.0
M) characteristic of proteins bound via hexahistidine tags.
As seen in Fig. 3B, both
and
G protein subunits as
well as the
3 integrin subunit coeluted with CD47 from
the 4N1K column, while none of these were recovered from the control
His6-4NGG column. To examine the mode of association of the
G proteins with CD47 immobilized on the 4N1K column, the lysate-charged
column was eluted sequentially with GTP
S or GDP
S, followed by
4N1K peptide (non-His-tagged) and then imidazole (1 M)
(Fig. 3C). GTP
S quantitatively eluted the
Gi
protein (and G
; not shown) while
GDP
S did not, the Gi
remaining on the column until
elution with 4N1K peptide. In addition, AlF4 also cleanly
eluted the Gi
protein (Fig. 3C), indicating that the association of the G proteins with the CD47 complex is released by activation of Gi
, a property shared with
receptor-G protein interactions. Interestingly, GTP
S and
AlF4, but not GDP
S also eluted about half of the CD47
from the 4N1K affinity column (not shown), suggesting that the
conformational change in the G
subunit associated with
activation is translated into a reduced affinity of CD47 for its ligand
on the column.
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Fig. 3.
Affinity chromatography of CD47-G protein
complexes. A, G and CD47 coelute. The
detergent-soluble fraction of C32 membranes was applied to a
nickel-His6-4N1K column as described under "Experimental
Procedures." After washing, the column was eluted with the indicated
concentrations of imidazole, and the fractions were analyzed by
SDS-PAGE and Western blotting with anti-G
and CD47 antibodies. The
detergents used were as follows: 30 mM
octyl-
-D-glucopyranoside (top
row); 1% w/v Triton X-100 (two bottom
rows). B, the complex contains G
,
G
, CD47, and integrin. The Triton X-100-soluble fraction
from C32 membranes or platelets was adsorbed on His6-4N1K
or His6-4NGG columns, washed with 50 column volumes of
HEPES-buffered saline-Triton buffer, and eluted with 1 M
imidazole. Fractions of the eluates were run on SDS-PAGE, blotted, and
probed with antibodies against G
, G
,
CD47, and
3 integrin. The peak fraction of each eluate
is shown. C, Gi
is eluted with GTP
S and
ALF4 but not GDP
S. C32 membrane lysates were adsorbed to
His6-4N1K affinity columns as above, washed, and eluted
with GTP
S (30 µM), GDP
S (30 µM), or
AlF4 (30 µM) (lane 1*).
Subsequently, each column was eluted with 500 µM 4N1K
peptide (lane 2) and then with 1 M
imidazole (IMID; lane 3). After
SDS-PAGE, the blot was probed with anti-G
.
S and
AlF4, but not GDP
S or ATP (all at 60 µM),
significantly reduced the amount of CD47 associated with the 4N1K
beads. The concentration dependence of the effect of the GTP
versus the GDP analog is shown in Fig. 4B. The
specific inhibitory effect of GTP and AlF4 on agonist
peptide binding to CD47 was also apparent in the affinity labeling of
CD47 with 125I-4N1K peptide (Fig. 4C). Thus, two
very different methods both give evidence of a negative heterotropic
effect of GTP and AlF4 on agonist binding to CD47. These
results indicate that not only does the CD47-integrin complex include a
Gi-like heterotrimeric G protein, but there is a direct
coupling between CD47 and the G protein.
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Fig. 4.
Effect of nucleotides and aluminum fluoride
on the binding of CD47 to 4N1K. A, a Triton X-100 (1%
w/v)-soluble fraction of C32 membranes was incubated with
His6-4N1K-charged nickel-NTA beads (or
His6-4NGG control beads) in the presence of the indicated
nucleotides or AlF4, all at 60 µM. The beads
were rapidly washed, incubated with 125I-mAb 2D3 against
CD47 for 1 h, and counted in a -counter. The experiment was
repeated twice with similar results. B, using the same
method as in A, the concentration dependence of the effect
of GTP
S and GDP
S on the binding of CD47 to the
His6-4N1K beads was determined. This experiment was
repeated twice with identical results. C, a crude membrane
preparation of C32 cells was affinity-labeled with
125I-4N1K as in Fig. 1 (3, 4, 11) except that the indicated
nucleotides and AlF4 were present during the labeling
reaction at 300 µM. Con indicates no
additions, and 4N1K (second lane) was present in
a 50-fold molar excess during the labeling reaction to determine
specificity of labeling of CD47. Autoradiograms of two separate
experiments are shown.
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Fig. 5.
4N1K depresses intraplatelet cyclic AMP
levels. Human platelets (2 × 108/ml) were
incubated with or without pertussis toxin (PTX) at 200 ng/ml
for 2 h in the presence of 100 nM prostaglandin
E1. They were then treated in suspension without stirring
(to prevent aggregation) in the presence of the indicated stimuli for 1 or 15 min, and the level of intraplatelet cyclic AMP was determined.
4NGG and 4N1K were at 50 µM, and thrombin receptor
peptide (TRP) was at 5 µM. These
concentrations were determined to give maximal responses.
S Binding to Membranes--
Bona
fide G protein-coupled receptors are able to stimulate the exchange of
GDP for GTP on the G
subunit of heterotrimeric G
proteins. Thus, we tested the ability of the CD47 agonist 4N1K to
stimulate GTP
35S binding to membranes prepared from K562
cells, which express relatively large amounts of CD47 and
v
3 (13). As seen in Fig. 6A, the CD47 agonist peptide
4N1K, but not the biologically inactive control peptide 4NGG
(KRFYGGMWKK), stimulated the rate of association of the radiolabeled
GTP analog with membranes. The stimulation of GTP
35S
binding by the agonist peptide occurs during the early part of the time
course as observed for other G protein-coupled receptors (20).
Gi is quite abundant in these cells (not shown), thus accounting for the robust, ~100% stimulation of the extent of GTP
binding by 5 min. The stimulation of GTP
35S binding is
concentration-dependent with maximal stimulation at 50 µM 4N1K, while 4NGG has no effect on binding at any
concentration (Fig. 6B).
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Fig. 6.
4N1K stimulates
GTP 35S binding to membranes.
A, membranes of
v
3-K562 cells
were incubated with the labeled GTP analog (~5 nM) and
either 4N1K or 4NGG (50 µM) at 30 °C as described
under "Experimental Procedures." At the indicated times, samples
were removed in triplicate, rapidly added to 3 ml of ice-cold buffer,
and filtered through nitrocellulose filters, which were washed with
another 3 ml of ice-cold buffer. The filters were dried and counted.
Background binding to the filters (no membranes) was less than 1000 cpm.
B, concentration dependence of the stimulation of GTP
binding by 4N1K. Assays were performed with
v
3-K562 membranes as in A with
an incubation time of 10 min at the indicated concentrations of 4N1K or
4NGG. C, the stimulation of GTP binding by 4N1K requires
CD47. Fibroblasts isolated from CD47-deficient (CD47
/
) mice (Ref.
9; F. Lindberg, unpublished results) were transfected with a CD47
expression construct or the empty vector as described by Lindberg
et al. (31). CD47 expression was confirmed by
fluorescence-activated cell sorting analysis. Membranes isolated from
the CD47-transfected and control cells as described under
"Experimental Procedures" were assayed for GTP
35S
binding as above using 50 µM 4N1K or 4NGG.
Lysophosphatidic acid (LPA; 10 µM) was used as
a positive control.
35S binding to about 30% the
level achieved with 4N1K in both CD47-deficient and CD47-containing
membranes (Fig. 6C, LPA). We also tested the effect of mastoporans, peptides reported to directly stimulate GTP
binding to G proteins, and observed only a minor increase in GTP
binding to K562 membranes (less than 10% of the increase seen with
4N1K, data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIb
3
activation (10). Other platelet receptors that costimulate
IIb
3 (e.g. thrombin, ADP, epinephrine, and thromboxane receptors) are 7TMS receptors coupled to
heterotrimeric Gi proteins (19). Activation of all of these costimulatory receptors in platelets leads to a precipitous drop in
intraplatelet cAMP levels, an event essential for further activation and aggregation (19). As shown in Fig. 5, activation of CD47 on
platelets results in an immediate and profound decrease in platelet cAMP.
1 (11) and
2 families (23,
24),2 as well as
3 (3, 4, 10). In all of these biological systems, the
effects of the CD47 agonists 4N1K and TSP-1 on cell spreading,
chemotaxis, and adhesion are blocked by pertussis toxin treatment of
the cells in question (4,
10).2,3 Thus, it appears that
different integrin
heterodimers associating with CD47 can all
couple to Gi protein-dependent pathways. This combinatorial diversity of the integrins that can associate with and be
regulated by CD47 may underlie some of the long noted, yet poorly
explained, variety of functions attributed to TSP proteins (1, 2). The
agonist peptide sequence RFYVVMWK is extremely well conserved in all
five TSP isoforms; thus, CD47 is probably a receptor for all TSP family members.
35S binding to membranes (Fig. 6). These
membranes contain few means of generating a signal intermediate between
CD47 ligation and Gi activation, since trisphosphate kinase
substrates and energy-requiring reactions are eliminated. Last, a
direct link between CD47 and Gi is indicated by the
negative heterotropic effect of G protein activators (GTP
S and
AlF4) on the binding of 4N1K to CD47.
-adrenergic receptor results in
disconnected transmembrane segments fully capable of signaling (27). In
addition, a functional
2-adrenergic receptor was
assembled by expression of two separate proteins, one containing
transmembrane segments 1-5 and the other containing segments 6 and 7 (28). (iv) Structural features conserved across the large 7TMS receptor
superfamily have some homologs in integrin-CD47 heterotrimers. These
include the DRY sequence, which occurs at the membrane-cytoplasmic
boundary of the third transmembrane segment of many 7TMS receptors, the
WXXXL sequence in transmembrane segment IV, and positively
charged residue clusters in cytoplasmic domains involved in G protein
coupling (29). Further experiments employing mutagenesis strategies are
under way to map the sites of G protein coupling in CD47 and thus test
this hypothesis. (v) The CD47 agonist peptide 4N1K is similar in
sequence to peptide ligands for other G protein-coupled receptors. For
example, the neurokinin I receptor, which can couple to Gi,
binds short peptide ligands (30). Among them is physalaemin with the
sequence -KFYGLM, which is similar to the sequence -RFYVVM of
4N1K/TSPs. Taken together, these considerations suggest that a
functional heterotrimeric 7TMS unit could be assembled from the
-
and
-chains of an integrin associated with the five TM segments of
CD47. This model is currently being tested.
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ACKNOWLEDGEMENTS |
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We thank Dr. Susanne Mumby and Dr. Patrick Casey for providing antibodies and Mark Crankshaw and the staff of the Washington University Protein and Nucleic Acid Chemistry Laboratory for peptide synthesis and purification. We thank Drs. Ken Blumer and Linda Pike for critical reading of the manuscript and many helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM51466 (to M. E. L.), GM54390, and CA65872 (to W. A. F.), GM38330 (to E. J. B.), and grants from Monsanto/Searle (to W. A. F., F. P. L., and E. J. B.).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.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biophysics, Box 8231, Washington University School of
Medicine, St. Louis, MO 63110. Tel.: 314-362-3348; Fax: 314-362-7183;
E-mail: frazier{at}biochem.wustl.edu.
2 W. A. Frazier, A.-G. Gao, J. Dimitry, J. Chung, E. J. Brown, F. P. Lindberg, and M. E. Linder, unpublished data.
3 X. Q. Wang and W. A. Frazier, manuscript in preparation.
4 J. Chung and W. A. Frazier, unpublished data.
5 W. A. Frazier, A.-G. Gao, J. Dimitry, J. Chung, E. J. Brown, F. P. Lindberg, and M. E. Linder, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
TSP, thrombospondin;
IAP, integrin-associated protein;
mAb, monoclonal antibody;
PAGE, polyacrylamide gel electrophoresis;
NTA, nitrilotriacetic acid;
7TMS, seven-transmembrane segment;
GDPS, guanyl-5'-yl thiophosphate;
GTP
S, guanosine 5'-3-O-(thio)triphosphate.
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REFERENCES |
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