(Received for publication, October 23, 1995; and in revised form, November 8, 1995)
From the
The C-terminal ``cell-binding domain'' (CBD) of
thrombospondin-1 (TS1) is a binding site for many cell types.
Cell-binding peptides based on the sequence RFYVVM from the CBD of TS1
affinity label a 52-kDa cell surface glycoprotein, which we show is
integrin-associated protein (IAP or CD47). IAP associates with
and thereby modulates the activity
of several integrins. Cells that express IAP bind strongly to TS1, the
CBD, and its active cell-binding peptides while IAP negative cells do
not. The 52-kDa protein is affinity labeled on IAP-positive but not
IAP-negative cells, and monoclonal antibodies against IAP specifically
immunoprecipitate the affinity-labeled 52-kDa protein from lysates of
IAP-positive cells. Consistent with the association of IAP with
integrin, the labeled 52-kDa protein
is immunoprecipitated by an anti-
antibody. Endothelial cells exhibit chemotaxis toward TS1 (at
concentrations above 10 nM) and RFYVVM peptides. Chemotaxis to
both agents is specifically inhibited by a function blocking anti-IAP
monoclonal antibody. These data establish IAP (CD47) as a receptor for
the CBD of TS1 and suggest a mechanism for the well established effects
of the CBD on cell motility.
The thrombospondins (TS) ()are a family of proteins
implicated in the regulation of the motility, proliferation, and
differentiation of many cell types(1, 2) . For
example, thrombospondin 1 (TS1) blocks angiogenesis by inhibiting the
chemotaxis and proliferation of endothelial cells and promoting their
differentiation into capillaries(1, 3) . These effects
of TS1 on cells are mediated through the binding of a number of domains
and peptide sequences of TS to several classes of cellular receptors
including heparan sulfate proteoglycans, sulfatides, CD36 (platelet
glycoprotein IV or IIIb), and a number of integrins of the
3 and
1 families (1, 2, 4) . Platelet
thrombospondin (TS1) is the prototypic member of this family, which now
consists of five isogenes and encoded isoforms (2, 4) . We have mapped cell-binding sites to peptide
sequences within seven of the eight domains of
TS1(5, 6, 7, 8) , including the
C-terminal ``cell-binding domain''
(CBD)(6, 7) , first identified as such with monoclonal
antibody (mAb) C6.7, which inhibits secretion-dependent platelet
aggregation(8, 9) , the attachment of many different
cell types to TS1(10, 11) , the chemotaxis toward TS1
of monocytes, polymorphonuclear monocytes(12) , tumor cells (13) , and smooth muscle cells (14) and a
Ca
influx stimulated by TS1(15) .
Given the important functional role of the CBD in TS1-cell interactions, we sought to determine the essential features of the CBD for cell binding and the identification of the receptor(s) through which the CBD exerts its effects on cells. Expression of recombinant CBD demonstrated that it contained a unique cell-binding site(s) independent of other sequences in TS1, including the RGD sequence, which is not contained within the CBD(6) . Using overlapping synthetic peptides (30-mers), we identified two homologous cell-binding sequences: RFYVVM within the fourth peptide (C4) and FIRVVM within the seventh (C7)(7) . The RFYVVM sequence is highly conserved in all TS family members(2, 7) . The C4- and C7-derived peptides competed with one another in cell adhesion assays suggesting that they bound a common receptor. Using derivatives of these peptides we affinity-labeled a 52-kDa protein on K562 cells (16) and many other cells and tissues. All of the properties of this hydrophobic, cell surface glycoprotein were consistent with those of a receptor for the CBD of TS1.
Integrin-associated protein (IAP) was first
identified by Brown and co-workers (17, 18) due to its
association with integrin purified
from placenta. IAP is expressed on many mammalian cells, has an M
of
50 kDa, and consists of an N-terminal
(extracellular) IgG variable type domain followed by five potentially
membrane-spanning, hydrophobic helices(18, 19) . IAP
was recently shown to be identical to the 1D8 antigen and CD47, which
is reduced on Rh
erythrocytes(19) . IAP
appears to be involved in signal transduction by
and
perhaps other integrins since mAbs directed to IAP block
integrin-stimulated phagocytosis(20) , an entactin-stimulated
oxidative bust in neutrophils and monocytes(21) , the inward
calcium current in endothelial cells (22, 23) , and
fibroblasts (15) adhering to vitronectin or fibronectin. The
wide expression of IAP suggests that it may have a general role in
integrin-mediated signal
transduction(17, 21, 24, 25) ;
however, studies of the IAP mechanism and its physiological role have
been hampered by the fact that its natural ligand is unknown. The
biochemical properties of IAP precisely mirror those found for the
52-kDa protein affinity-labeled by the CBD peptides. We report here
that IAP is a receptor for the TS1 CBD and its VVM-containing peptides
and that a function-blocking anti-IAP mAb inhibits the chemotactic
response to TS1 and its CBD peptides in endothelial cells.
Monoclonal antibodies LM609
(anti-) and P1F6
(anti-
) were the generous gifts of
Dr. D. Cheresh, Scripps Research Institute. B6H12 and 2D3, anti-human
IAP mAbs, have been previously
described(17, 18, 19, 20, 21) .
OV10 cells are a subclone of OVM1 human ovarian carcinoma (kindly
provided by Dr. W. H. Stimson, Strathclyde University, Glasgow), which
fail to express IAP. They were transfected with pIAP45(18) , an
IAP expression construct, and IAP-positive clones were selected by
fluorescence-activated cell sorting. K562 cells expressing
are described in Blystone et al. (24). Human platelet TS1 was purified as described(8) ;
the rCBD was expressed as a His
-tagged protein in Escherichia coli using pQE30 (Qiagen) and purified by
nickel-nitriloacetate chromatography. Peptides were synthesized,
purified, and characterized by the Washington University Protein and
Nucleic Acid Chemistry Laboratory as
described(7, 16) .
Cell adhesion assays were
performed and quantified with endogenous cellular phosphatase activity
as described in Prater et al.(5) . Affinity labeling
of cells and detergent lysates with I-4N1K peptide
(KRFYVVMWKK) was performed as described in Gao and
Frazier(16) . Immunoprecipitations employed the indicated
primary mouse mAb and anti-mouse IgG-agarose (Sigma) and were done as
described in Brown et al.(17) . Iodinated samples were
dissolved in boiling SDS sample buffer with reduction and
electrophoresed on 10% SDS gels, which were dried and autoradiographed (16) . Chemotaxis assays were performed in modified Boyden
chambers (Neuroprobe, Cabin John, MD) using 8.0-µm pore size
gelatin-coated polycarbonate filters as described by Tolsma et
al.(26) . Antibodies (purified IgG) were added to the
cells at the indicated concentrations before placing the cells into the
wells of the chamber.
To test whether IAP was necessary for the binding of cells to
the CBD, we first had to find a cell line deficient in IAP expression
to serve as a negative control. Previous studies with anti-IAP mAbs (17, 18, 19, 20, 21, 28) indicated
that IAP has an extremely broad cell and tissue distribution. The
ovarian carcinoma cell line OVM1 was found to have low levels of IAP
expression, and a subclone designated OV10 expressed essentially no IAP
by flow cytometric and Western blotting criteria. ()Suspensions of OV10 (IAP negative) and OV10
(IAP transfected) cells were incubated in plastic 96-well plates
coated with intact TS1, rCBD, and three versions of the C4 peptide.
Fibronectin-coated wells served as a positive control (OV10 cells
express
) and bovine serum
albumin-coated wells as the negative control. Fig. 1shows that
both OV10 and OV10
cells adhered equally well to
fibronectin-coated wells, but twice as many OV10
cells
expressing IAP adhered to TS1 as IAP-deficient OV10. Adhesion to the
rCBD and all three C4 peptides showed an even greater preference for
the IAP-expressing cells. Like most cells, OV10s probably express
receptors for several domains of intact
TS1(1, 2, 4) . Both cell lines adhered poorly
to peptides from the calcium-binding domain of TS1 (CaIII and CaVII),
although, interestingly, the OV10
cells adhered better
to CaVII, which contains the RGD sequence of TS1. This may be due to an
effect of IAP expression on the function of the low levels of
expressed by OV10 cells (not shown).
Peptide Mal II is a cell-binding peptide from the type 1 repeats of
TS1(5) , and both cell types adhered to it to the same extent.
Thus, expression of IAP confers on the OV10
cells the
ability to adhere to the VVM peptide site(s) within the CBD of TS1.
Figure 1:
Adhesion
of OV10 cells to TS1, CBD, and its peptides. IAP-negative,
vector-transfected OV10 cells and pIAP45-transfected OV10 IAP positive cells were allowed to adhere for 90 min to plastic
wells coated with the indicated proteins and peptides. The wells were
washed and adhesion-quantified by endogenous phosphatase activity with p-nitrophenyl phosphate as a substrate(5) .
,
vector-transfected OV10 cells;
, IAP-transfected OV10 cells.
Peptides are: C4, RFYVVMWKQVTQSYWDTNPTRAQGYSGLSV; 4N1,
RFYVVMWKQVTQSYWDTN; 4NK, KRFYVVMWKQVTQSKKY; CaVII,
KDNCRLVPNPDQKDSDGDGRGDACK; CaIII, NCPYNHNPDQADTDNNGEGDAC; Mal II,
SPWSSASVTAGDGVITRIR. FN, fibronectin; TSP, thrombospondin.
To determine if IAP was acting directly as the CBD receptor, we
affinity-labeled cells with peptide 4N1K, a derivative of the C4
peptide, with the sequence KRFYVVMWKK. This peptide has been shown to
specifically label a 52-kDa plasma membrane protein on intact cells and
in detergent lysates(16) . When OV10 cells were
affinity-labeled with 4N1K, no labeled proteins were found (Fig. 2, lanes 3 and 4). In contrast, labeling
of OV10 cells revealed a 52-kDa protein along with the
previously characterized 37-kDa proteolysis product (lane 1).
Affinity labeling of both bands could be inhibited completely by excess
cold peptide, demonstrating the specificity of labeling (lane
2). These bands are identical to those labeled on K562 cells (lanes 5 and 6). Immunoprecipitation of labeled OV10
cell lysates with two anti-IAP mAbs revealed no 52-kDa protein (lanes 7 and 8) while the labeled protein was
abundant in anti-IAP precipitates of OV10
cells (lanes 9 and 10) but not in control anti-HLA (lane 11) or anti-TNF receptor (lane 12)
precipitates. Thus in IAP-negative OV10 cells, transfection of IAP
confers binding to the CBD, and IAP is specifically labeled by the CBD
peptide. These data demonstrate a direct interaction of IAP with the
CBD. The direct binding of the CBD peptides to IAP is confirmed by the
observation that the extracellular IgGv domain of IAP, when expressed
as an Fc fusion protein, (
)binds specifically to the cell
binding VVM containing C4 and C7 peptides but not to six other 30-mers
representing the rest of the CBD sequence (data not shown) (27) .
Figure 2:
Affinity labeling of IAP with CBD peptide.
Peptide I-KRFYVVMWKK (4N1K) was used to affinity label
intact OV10
cells expressing IAP (lanes 1 and 2), parental OV10 cells (OV10
) (lanes 3 and 4), and K562 cells (lanes 5 and 6)
as described by Gao and Frazier(16) . Unlabeled 4N1K peptide
was present in reactions for lanes 2, 4, and 6.
Detergent lysates of OV10
(lanes 7 and 8) or OV10
(lanes 9-12) cells
were affinity-labeled and then precipitated with the following mAbs:
anti-IAP B6H12 (lane 9); anti-IAP 2D3 (lane 10);
anti-HLA W6/32 (lane 11); anti-TNF receptor (lane
12). Detergent lysates of K562 cells expressing
(
-K562) were
labeled and precipitated with the following mAbs: B6H12 (lane
13); 2D3 (lane 14); W6/32 (lane 15); anti-TNF
receptor (lane 16); anti-
P1F6 (lane 17); and anti-
LM609 (lane 18).
Because IAP associates with
integrin(17) , we examined
4N1K labeling of IAP in
-transfected
K562 cells(20) . This cell line was used since OV10 cells
express little
(not shown). When a
detergent lysate was first labeled with 4N1K and then
immunoprecipitated with anti-IAP mAbs B6H12 (lane 13) or 2D3 (lane 14) the labeled 52-kDa IAP band was recovered in the
precipitate as with the OV10
cells. No labeled band
was recovered in precipitates of anti-HLA or anti-TNF receptor (another
55-kDa protein) or anti-
mAbs (lanes 15-17). Interestingly, immunoprecipitation of
labeled cell lysates with
antibody (lane 18) coprecipitated labeled IAP, suggesting that IAP can
interact simultaneously with
and the
CBD peptide.
The CBD of TS1 stimulates cell motility via haptotaxis,
chemotaxis, and chemokinesis as judged by inhibition of these processes
in several cell types by mAb
C6.7(2, 10, 12, 13, 14) .
We have previously reported that intact TS1 at concentrations above 10
nM could stimulate the chemotaxis of endothelial
cells(26) . We recently found that the CBD VVM-containing
peptides act as attachment factors for several types of endothelial
cells including HUVEC and that the C4 peptides affinity label a 52-kDa
membrane protein on these cells with properties identical to those
reported (16) for the protein from K562 cells. HUVECs express
high levels of IAP as determined by flow cytometry (not shown).
Furthermore, the CBD peptides are able to stimulate chemotaxis and
chemokinesis of endothelial cells. ()Thus the effects of
anti-IAP mAbs on chemotaxis to TS1 and the CBD peptide 4N1K were
determined using HUVEC as the test cell (Fig. 3). The anti-IAP
mAb B6H12, previously characterized as a function-blocking mAb (17, 20, 23, 28) , inhibited, in a
concentration-dependent way, chemotaxis of endothelial cells toward
both TS1 and the 4N1K peptide while 2D3, in other assays a nonblocking
anti-IAP mAb(17, 23, 28) , had no effect.
These data indicate that IAP is the CBD receptor responsible for the
stimulation of cell migration.
Figure 3: Effect of anti-IAP mAbs on endothelial chemotaxis. Endothelial (HUVEC) chemotaxis was stimulated by TS1 or the CBD peptide 4N1K in the presence of the indicated antibodies. Chemotaxis was performed as described by Tolsma et al.(26) . After staining, cells were counted in 5 high power fields (hpf) in each of the triplicate wells for each concentration of mAb. The number of cells migrating randomly in the absence of chemoattractant has been subtracted from all data points.
The data presented here identify IAP as a novel receptor for
the CBD of TS1 and probably other TS isoforms as well since the RFYVVM
sequence in peptide C4 of the CBD is highly
conserved(2, 7) . This conclusion is based on: (i) the
marked enhancement of OV10 cell adhesion to TS1, the rCBD, and the
CBD-derived peptides by transfection of OV10 cells with IAP; (ii)
affinity labeling of IAP with the 4N1K peptide; and (iii) inhibition of
TS1- and 4N1K-stimulated endothelial cell chemotaxis by a
function-blocking but not by a non-inhibitory anti-IAP mAb.
Interestingly, it seems that IAP, which is known to physically
associate with integrins(17) , can maintain
its integrin association even when bound to the CBD peptide, since
anti-
will immunoprecipitate
affinity-labeled IAP. This observation, along with the proximity of the
RGD sequence of TS1 to the CBD(1, 2, 6) ,
raises the intriguing possibility that TS1 can simultaneously engage
both IAP and its associated
integrin. This could
occur in an ordered fashion, thus providing a physiological mechanism
for the exposure of the normally cryptic RGD sequence of TS1 (29) and an explanation for the reports that
and platelet
can act as TS1
receptors(30, 31) .
Based on the inhibitory action
of mAb C6.7, the CBD of TS1 has been thought to stimulate the motility
of a number of cell
types(11, 12, 13, 14) . The C4
CBD-derived peptides are chemotactic for endothelial cells (this
paper). Blockade by an anti-IAP mAb establishes this
chemotactic response as a direct biological consequence of the
interaction of the CBD peptide with IAP. Two possible mechanisms for
IAP involvement in chemotaxis are suggested by other data. First, an
anti-IAP mAb has been shown to block the Ca
influx of
endothelial cells (23) adherent to RGD-containing substrates.
If the natural ligand, TS1, or its peptides were to stimulate
Ca
influx via IAP, this could provide a mechanism for
directed motility. That such a stimulation occurs has been shown by
Tsao and Mousa(15) , who found that TS1 and the C4 peptide
RFYVVMWK stimulated a transient Ca
influx in
fibroblasts, which was partially blocked not only by mAb C6.7 (in the
case of TS1) but also B6H12, the same function blocking anti-IAP mAb
used in our studies. These data confirm that a known function of IAP, i.e. stimulation of Ca
entry(23) ,
can be activated by the C4 peptide RFYVVMWK(15) .
The second
possible mechanism for stimulation of cell motility by IAP derives from
the association of IAP with and the
role of this integrin in the motility of cultured endothelial
cells(32) . Thus CBD binding to IAP may modulate the affinity
or avidity of
, which is used for
adhesion and traction on the gelatin-coated (RGD-containing) filter (32) . In this way the CBD or its C4 peptides, all of which
lack an RGD sequence, could act on
through binding to IAP. The
IAP-
association is also interesting
in light of recent data implicating
in angiogenesis where it acts, at least in part, by protecting
endothelial cells from apoptosis(33, 34) . TS1 plays
an important regulatory role in endothelial cells, where it promotes a
differentiated phenotype(1, 2, 3) and
antagonizes the initial events of angiogenesis(26) . It is
interesting to speculate that TS1-IAP interaction may modulate the
function of
during angiogenesis.
Moreover, the IAP-
complex has been
shown to regulate the function of other
integrins(19, 20, 21, 24, 25) .
It is possible that TS1 may exert many of its apparently diverse
biological effects(1, 2, 3, 4, 11, 12, 13, 14, 15) through
modulation of this signaling complex.