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From the
Received for publication, March 10, 2000, and in revised form, August 3, 2000
Lateral association between different
transmembrane glycoproteins can serve to modulate integrin function.
Here we characterize a physical association between the integrins
Integrins comprise a large family of Several classes of cell surface glycoproteins have been documented to
play a role in integrin-mediated events, the most widely studied being
the integrin-associated protein
(IAP1/CD47) and the unrelated
transmembrane 4 superfamily (TM4SF or tetraspannins). IAP associates
with the Other surface molecules have also been implicated in the modulation of
integrin-mediated functions. The widely expressed CD98 molecule
(initially described as an early T-cell activation antigen) was found
to regulate Another candidate co-associated regulator of integrin-function is the
transmembrane glycoprotein CD36. The archetype member of a small gene
family, CD36 is unrelated to integrins or to currently known
integrin-accessory molecules. CD36 expression is prominent on the
surface of platelets, capillary endothelial cells, macrophages, and
cultured cell lines from a variety of sources including melanoma (reviewed in Refs. 41 and 42). A study using chemical cross-linking demonstrated that CD36 is spatially associated with the
The phagocytic uptake of apoptotic cells by macrophage requires the
coordinate function of CD36 and the Possibly significant in this regard is the reported association of CD36
with plasma membrane microdomains or "rafts" enriched in
cholesterol, sphingomyelin, and glycolipids. Rafts can be isolated biochemically as low density Triton X-100-insoluble membranes, and such
isolates have been termed detergent-insoluble
glycosphingolipid-enriched complexes (DIGs), also described as DRMs,
GEMs, TIM, TIFF, and LDTI (reviewed in (53-55)). CD36 was found to be
highly enriched in DIGs from a variety of cellular sources including
lung endothelium (56), platelets (57), and transfected COS-7 cells
(58). Raft domains have been implicated in a variety of cellular
processes including signal transduction, membrane trafficking,
cholesterol homeostasis, the regulation of apoptosis, and cell motility
(53-55, 59, 60). The plasma membrane invaginations known as caveolae may represent a specialized structural form incorporating raft components contained within the structural coat protein caveolin (61).
Early studies suggested that integrins were not compartmentalized to
rafts/caveolae (56, 62), but subsequent reports have indicated that a
proportion of The present study was initiated to examine further the reported effect
of CD36 on melanoma cell morphology and growth (51). A melanoma cell
line that did not express endogenous CD36 was identified, and permanent
CD36 transfectants were isolated. No differences were found in the
morphology or growth rates of the transfectants compared with the
parent cell line, but the CD36-transfected cells displayed higher
haptotactic migration on extracellular matrix substrates. The
possibility that integrins might be implicated in this functional
effect was investigated by seeking a co-association between CD36 and
endogenous integrins on the melanoma cell surface. Co-immunoprecipitation studies revealed a physical association between
CD36 and the integrins Cell Lines and Antibodies--
The MV3 melanoma cell line (a
gift of Dr. G. N. P. van Muijen) was isolated from a lymph
node metastasis as described previously (68). C32 and WM115 melanoma
and COS-7 monkey kidney cell lines were obtained from the ATCC
(Manassas, VA). All cells were routinely cultured in Dulbecco's
modified Eagle's medium (CSL, Parkville, Australia) supplemented with
10% fetal bovine serum (Cytosystems, Castle Hill, Australia).
The monoclonal antibodies (mAbs) 13 and 16 directed against the
Construction of Expression Vectors and Cell
Transfection--
The human cDNAs for CD36 (a gift of Dr. A. W. Boyd) and ICAM-1 (gift of Dr. D. Simmons, University of Oxford,
Oxford, UK) in the pCDM8 vector were subcloned to the high level
eukaryotic expression vector pEF.BOS (71) using XbaI
restriction endonuclease digestion. To substitute the carboxyl-terminal
cytoplasmic tail of CD36 with the ICAM-1 cytoplasmic tail (CD36/ICAM
tail chimera) the pEF.BOS.ICAM-1 plasmid was used as template for
polymerase chain reaction amplification using oligonucleotides
5'-cGcAtGcctctataaccgccagcggaag-3' and 5'-ccccagggccatgcctcccagc-3',
which introduced an SphI restriction site to the 5'-end of
the product (mismatched oligonucleotide bases are represented in
uppercase type). Following digestion with
SphI/MscI, the resulting fragment was then
subcloned to an MscI/SphI partially digested
"vector" fragment of pEF.BOS.CD36, thereby inserting the 31-amino
acid ICAM-1 cytoplasmic tail following CD36 cysteine residue 466. A
chimera consisting of the amino terminus of ICAM-1 and the CD36
carboxyl-terminal transmembrane region and cytoplasmic tail
(ICAM/CD36) was prepared by digestion of pEF.BOS.CD36 and
pEF.BOS.ICAM-1 plasmid DNA with HincII/MscI and MscI restriction endonucleases, respectively. The
MscI fragment encoding the 5'-sequence of ICAM-1 and the
HincII/MscI fragment encoding the 3' sequence of
CD36 were ligated together, resulting in the ICAM-1 extracellular
region amino acids 1-409 fused to residues 390-472 of CD36. To
manufacture an amino-terminal truncation of CD36 with the FLAG
epitope tag placed at the N terminus (FN
Stable transfections of MV3 cells were performed using the
LipofectAMINE reagent (Life Technologies) according to the
manufacturer's instructions. A 20:1 excess of the pEF.BOS vector
(which lacks a eukaryotic selection marker) to pREP9 (encoding neomycin
phosphotransferase; Invitrogen, San Diego, CA) was used, followed by
selection with 500 µg/ml G418 (Life Technologies). This procedure
facilitated a high level of expression of CD36, negating the need for
dilution cloning. For transient expression, MV3 and COS-7 cells were
electroporated (300 V/250 microfarads using a Bio-Rad Gene Pulser) with
150 µg/ml plasmid DNA in Hebs buffer (20 mM Hepes, pH
7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM
Na2HPO4, 6 mM dextrose), and the
cells were analyzed 2-4 days post-transfection.
Flow Cytometry--
Cells were harvested using trypsin-versene
solution (CSL) and washed in phosphate-buffered saline (PBS). The
respective primary mAbs were then incubated at 4 °C for 30 min,
after which the cells were washed twice with PBS and the bound mAb was
detected by incubation with fluorescein isothiocyanate-conjugated sheep
anti-mouse immunoglobulin (F(ab)2 fragment; Silenus,
Dandenong, Australia). Cells were washed again; fixed using 2% (w/v)
glucose, 1% (w/v) formaldehyde, 0.02% (w/v) azide in PBS; and
analyzed on a Becton Dickinson (Mountain View, CA) FACScan.
Cell Adhesion and Haptotactic Cell Migration Assays--
Cell
adhesion to extracellular matrix proteins (collagen type I,
fibronectin, or laminin; Sigma) was measured as described previously (72). For migration assays, the underside of replicate Transwell migration inserts (8-µm pore size, 6.5-mm diameter; Costar,
Corning, NY) were coated for 60-90 min at 37 °C with the indicated
matrix proteins (20 µg/ml) or with bovine serum albumin (BSA; 0.5%
(w/v)) in 200 µl of PBS. The wells were washed once with migration
buffer (serum-free medium supplemented with 0.5% BSA), and 500 µl of
migration buffer was added to the bottom chamber. Cells were detached
using trypsin-EDTA (0.25% (w/v) and 5 mM, respectively)
and washed three times in migration buffer before seeding to the top of
the filter (105 cells in 100 µl). After 24 h at
37 °C, adherent cells were detached using trypsin/EDTA from the
upper and lower membrane surfaces and separately pooled with the
supernatants collected from each respective chamber. Cells were counted
on a CASY-1 Cell Analyser (Scharfe Systems GmbH, Reutlingen), and cell
migration was calculated as the percentage of cells harvested from the
underside of the filter divided by the total number of cells counted.
All assays were performed on triplicate wells and repeated several times.
Cell Surface Labeling and Immunoprecipitation--
A modified
lactoperoxidase method was used for cell surface radiolabeling with
Na125I (Australian Radioisotopes, Lucas Heights, Australia)
as described previously (23). For biotin labeling of cell surface
antigens, cells were resuspended in 10 mM sodium
tetraborate, pH 8.8, 150 mM NaCl containing 50 µg/ml
biotinamidocaproate N-hydroxysuccinimide ester (Sigma) and
incubated for 15 min at room temperature before quenching of the
reaction by the addition of NH4Cl to a 10 mM final concentration. After washing with PBS,
125I/biotin-labeled cells were lysed for 60 min at 4 °C
with Brij 96 lysis buffer (1% polyoxyethylene 10 oleyl ether (Sigma)
in 25 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl2 supplemented with protease inhibitors
(2 mM phenylmethylsulfonyl fluoride, 10 µg/ml soybean
trypsin inhibitor, and 2.5 mM iodoacetamide (all from Sigma)). Insoluble material was then removed by centrifugation for 10 min at 14,000 × g. The lysates were precleared twice using protein A beads (Amersham Pharmacia Biotech) and/or rabbit anti-mouse immunoglobulins (DAKO, Carpentaria, CA) coupled to CNBr-activated Sepharose beads (Amersham Pharmacia Biotech) in combination with human
transferrin (Calbiochem) coupled beads for 1-2 h at 4 °C. Lysates
were then incubated for 2 h (biotin) or overnight
(125I) with either mAb combined with rabbit anti-mouse
immunoglobulin-coupled beads, with pAb with protein A beads (indirect
capture), or with mAb directly coupled to beads (as described above).
Because CD36 was poorly represented in cell surface immunoprecipitates
relative to
Where samples were subjected to secondary immunoprecipitation, the
beads were first washed with lysis buffer, and the antigen(s) were
eluted from the beads using acidic buffer (50 mM
glycine-HCl, pH 2.5, 0.15 M NaCl, 0.1% Triton X-100). The
eluate was immediately neutralized with 2 M Tris, pH 8.0, passed through a PD10 column (Sephadex G25M; Amersham Pharmacia
Biotech), and eluted in 1% Triton X-100 containing lysis
buffer. The eluate was then subjected to a second round of
immunoprecipitation and processed as described above.
Immunoblotting--
Cells were lysed and immunoprecipitated as
described above. Samples were resolved by SDS-PAGE and transferred to
nitrocellulose using 25 mM Tris, 192 mM
glycine, 20% methanol transfer buffer. Nitrocellulose membranes were
blocked in 5% skim milk in TTBS (20 mM Tris, pH 7.5, 500 mM NaCl, 0.05% Tween 20) before incubation with specific
antibodies in 1% skim milk TTBS. Membranes were then incubated with
horseradish peroxidase-conjugated goat anti-mouse or rabbit
immunoglobulins (Bio-Rad), and horseradish peroxidase complexes were
detected by the Renaissance chemiluminescence system (PerkinElmer Life Sciences).
Immunofluorescent Cell Staining and Confocal Microscopy--
MV3
cells were harvested as described above and allowed to attach to glass
coverslips overnight. Following fixation with 4% formaldehyde (w/v) in
PBS for 20 min at room temperature, the coverslips were washed in 0.1%
BSA, PBS (PBSA) and permeabilized with Triton X-100 (0.1% (v/v) in
PBSA) for 10 min at room temperature. Antibodies (diluted in PBSA) were
then added overnight at 4 °C, and the coverslips were washed four or
five times in PBS before mounting in Mowiol 4-88 (0.1 g/ml;
Calbiochem) dissolved in a mixture of 12 ml of Tris-HCl (pH 8.5), 6 ml
of Citifluor AF1 (Citifluor Ltd., London, UK), and 6 ml of distilled
water. Fluorescein isothiocyanate-conjugated anti-integrin mAbs
together with an anti-CD36 (11H5) TAMRA conjugate (prepared according
to the manufacturer's recommended protocol; Molecular Probes, Inc.,
Eugene, OR) were used for the two-color analysis. Cells were examined
by laser-scanning confocal microscopy (Zeiss LSM510, Zeiss Microscopes,
Welwyn, UK).
Detergent-insoluble Glycosphingolipid-enriched Membrane Raft
Domains--
DIGs were prepared by floatation in sucrose density
gradients as described previously (58). Briefly, whole cell lysates solubilized in 1% Triton X-100 in MES-buffered saline (pH 6.5) were
adjusted to 40% sucrose and applied under a discontinuous 5-30%
sucrose gradient. Following ultracentrifugation, DIGs were found to
separate as a low density light-refractive band. Twelve fractions were
collected from the top of the gradient, and equal protein amounts of
each were analyzed by SDS-PAGE and immunoblotting (protein
concentrations were determined using the BCA assay; Pierce). Densitometric analysis was performed on nonsaturating exposures of
immunoblot films. Images were captured using a UMAX powerlook II
scanner, and densitometric quantitation was performed using the NIH
Image software package, version 1.6.1 (written by W. Rasband, National
Institutes of Health). Enrichment in DIGs was calculated as the total
antigen density in DIG-containing fractions divided by the total
antigen density detected across the gradient (percentage of antigen in
DIGs).
The Integrins
Next we examined the transfected cells for a physical association
between CD36 and integrins, since these molecules provide the
mechanical elements of cell migration. The transfected MV3 cells were
surface-labeled with radioiodine and lysed with Brij 96 detergent, and
the soluble cell lysates were subjected to immunoprecipitation for CD36
or
To determine whether this was the case in melanoma cells expressing
endogenous CD36, the same immunoprecipitation experiments were carried
out with lysates of surface radioiodinated C32 and WM115 melanoma
cells. As before, CD36 antibodies co-precipitated labeled bands in the
region of
To identify specific integrin(s) associating with CD36, reprecipitation
assays were employed. Phenotyping of the MV3 cells by flow cytometry
demonstrated that they expressed each of the
The co-association was then confirmed by immunoblotting (Fig.
3D). MV3.CD36 cells were lysed in Brij lysis buffer and
immunoprecipitated for CD36 and for
To confirm that the association between the Association with Integrins Requires the Extracellular Domain of
CD36--
To elucidate the nature of the CD36/
The MV3 cells were then transfected with an ICAM/CD36 chimera, encoding
the extracellular domain of ICAM-1 and the CD36 carboxyl-terminal transmembrane/cytoplasmic sequences (Fig. 5A), and the cell
lysates were precipitated with an anti-ICAM-1 mAb. This antibody was
found to precipitate endogenous ICAM-1 from the control transfected MV3
cells (Fig. 5D, BOS), and no integrin bands were seen to
co-precipitate with the ICAM-1 molecule. In cells transfected with the
ICAM/CD36 construct, the chimeric molecule was found to co-precipitate
with endogenous ICAM-1, but again there were no integrin bands
identified in the precipitate (Fig. 5D). Therefore, since no
association was seen when the extracellular region of CD36 was replaced
with ICAM-1, although the transmembrane domain and cytoplasmic tail of
CD36 were left intact, these data suggest that the association between
CD36 and integrin molecules occurs extracellularly.
These findings established that the association between the
To investigate this, a molecule was constructed lacking the
amino-terminal and cytoplasmic domains; this construct was tagged with
a FLAG epitope and incorporates a cleavable signal sequence (FN Expressed CD36 Sequesters a Proportion of
MV3.BOS and MV3.CD36 transfected cells were lysed with 1% Triton X-100
in MES-buffered saline buffer and subjected to floatation on
sucrose gradients as described under "Experimental Procedures." Following fractionation of the gradient, equal protein amounts from
each fraction were separated on SDS-PAGE, and the distribution of
various antigens was analyzed by immunoblotting. As expected, CD36 was
not detected in the BOS-transfected cells, but, following CD36
transfection, it was found to be greatly enriched in the fractions
representing the light-refractile DIG band (Fig.
7, compare A and
B). Control and CD36-transfected lines displayed abundant
caveolin, also concentrated in the DIG fractions. The same fractions
were also probed for an ER resident protein, calreticulin (82), and, as
expected, this did not co-migrate with the DIGs but was primarily
confined to the fractions of the gradient corresponding to soluble
proteins (Fig. 7, A and B). The
In these analyses (Fig. 7C), the proportion of the label for
each marker in the DIG fractions was measured as a fraction of the
total antigen density across the gradient (see "Experimental Procedures"). Both CD36 and caveolin were contained primarily in the
DIG fractions, and the proportion of caveolin in these fractions was
the same in both transfected cell populations. Similarly, the ER
resident protein, calreticulin, was found across the soluble fractions with little protein identified in DIGs; this distribution also did not alter after CD36 transfection. In contrast, the proportion of mature
These results suggest that CD36 can sequester a proportion of
It is increasingly being recognized that integrin function can be
modulated by association with other transmembrane glycoproteins, as
well as by cytosolic proteins, and several such integrin-associated glycoproteins have been identified. To this list can now be added CD36.
As demonstrated here in melanoma cells, CD36 forms a physical association with the The specific integrins identified as associating with CD36 were
An important finding in the present study was that Integrins co-precipitate with endogenous and transfected uPAR in a
complex that also contains caveolin (40, 83). Caveolin itself can also
bind cholesterol, Src family kinases, heterotrimeric G proteins, and
Ha-Ras (84, 85), thereby contributing to the formation of
caveolae rich in signaling molecules. It might be considered therefore
that by associating with Precisely how CD36 might regulate integrin function remains to be
determined, but several scenarios can be envisioned. By the recruitment
of adjacent integrins into membrane microdomains together with
Src-related kinases, CD36 could serve to promote integrin-mediated
signaling. Further, membrane microdomains are highly enriched in
gangliosides (80), which themselves can regulate protein kinase
function (92) and, more particularly, have been shown physically to
associate with Arg-Gly-Asp-directed integrins and to be involved in
their adhesive function (72, 93, 94). Not necessarily unrelated to such
a role in sequestration is the possibility that CD36 can function as an
accessory molecule, complementing integrin ligation with certain
extracellular matrix ligands. Precedents for integrin accessory
molecules regulating or stabilizing the adhesive function of associated
integrins include uPAR, which contains a vitronectin binding site and
can regulate the relative binding of Finally, at the initiation of this study, we expected to find an
association between CD36 and We thank all those investigators mentioned
who supplied reagents essential to this work. We are grateful to Dr.
Eric Brown (Division of Infectious Diseases, Washington University, St.
Louis) for critically reviewing a draft version of the manuscript and for valuable suggestions.
*
This work was supported by the National Health & Medical
Research Council (Australia).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.
§
Supported by an Australian Postgraduate Award scholarship. To whom
correspondence should be addressed: Cancer Research Unit, Level 5, David Maddison Clinical Sciences Building, Cnr. King & Watt St.,
Newcastle, NSW, Australia 2300. Tel.: 61-2-49236843; Fax:
61-2-49236984; E-mail: rthorne@mail.newcastle.edu.au.
Published, JBC Papers in Press, August 23, 2000, DOI 10.1074/jbc.M003969200
2
R. F. Thorne and G. F. Burns,
unpublished data.
3
R. G. Parton, R. F. Thorne, and G. F. Burns,
unpublished data.
The abbreviations used are:
IAP, integrin-associated protein;
DIGs, detergent-insoluble
glycosphingolipid-enriched complexes;
TM4SF, transmembrane 4 superfamily;
mAb, monoclonal antibody;
pAb, polyclonal antibody;
ICAM-1, intercellular adhesion molecule 1;
PBS, phosphate-buffered
saline;
PAGE, polyacrylamide gel electrophoresis;
uPAR, urokinase
plasminogen activator receptor;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
MES, 4-morpholineethanesulfonic acid;
BSA, bovine serum albumin.
The Integrins
3
1 and
61 Physically and Functionally Associate
with CD36 in Human Melanoma Cells
REQUIREMENT FOR THE EXTRACELLULAR DOMAIN OF CD36*
§,
,
Cancer Research Unit and Department
of Microbiology, Faculty of Medicine and Health Sciences, University of
Newcastle, New South Wales 2308, Australia, the ** Department of
Respiratory Medicine, John Hunter Hospital, Newcastle, New South Wales
2305, Australia, and the ¶ Richard Dimbleby Department of Cancer
Research/Imperial Cancer Research Fund, Rayne Institute, St Thomas'
Hospital, London SE1 7EH, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1 and
61 and CD36 on the surface of melanoma
cells and show that ectopic expression of CD36 by CD36-negative MV3
melanoma cells increases their haptotactic migration on extracellular
matrix components. The association was demonstrated by
co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of
surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy
illustrated the co-association of 3 and CD36 in cell
membrane projections and ruffles. A requirement for the extracellular
domain of CD36 in this association was shown by co-immunoprecipitation
experiments using surface-labeled MV3 melanoma or COS-7 cells that had
been transiently transfected with chimeric constructs between CD36 and
intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant
of CD36. CD36 is known to engage in signal transduction and to localize
to membrane microdomains or rafts in several cell types. Toward a
mechanistic explanation for the functional effects of CD36 expression,
we demonstrate that in fractionated Triton X-100 lysates of the MV3
cells stably transfected with CD36, CD36 was greatly enriched with the
detergent-insoluble fractions that represent plasma membrane rafts.
Significantly, when these fractionated lysates were reprobed for
endogenous 1 integrin, it was found that a 4-fold
increase in the proportion of the mature protein was contained within
the detergent-insoluble fractions when extracted from the
CD36-transfected cells compared with MV3 cells transfected with vector
only. These results suggest that in melanoma cells CD36 expression may
induce the sequestration of certain integrins into membrane
microdomains and promote cell migration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
heterodimeric
transmembrane proteins that function as key receptors for cellular
attachment to the extracellular matrix and to cellular ligands (1).
Integrin function extends beyond cell adhesion per se, and
integrin-mediated signaling influences a range of biological processes
including cellular division and migration, differentiation, and
apoptosis (reviewed in Refs. 1-5). Integrin function is complex and is regulated at many levels. The binding of ligand to integrins can initiate signaling (1, 2), with bidirectional "cross-talk" occurring between integrins and other pathways (e.g. Refs. 6 and 7), including the functional modulation of other integrin molecules
(8-10). Much emphasis has also focused on the process of
"inside-out" signaling, where intracellular events enacted through
the relatively short cytoplasmic tail sequences of integrins have a
profound influence on the extracellular function of the integrin (1).
In addition to the molecular interactions occurring between cytoplasmic
proteins and the cytoplasmic domains of integrins (reviewed in Refs. 11
and 12), emerging evidence indicates that a number of surface
transmembrane glycoproteins can associate with integrins and modulate
their cellular functions (12).
v3 integrin (13) and influences
integrin-mediated signal transduction, phagocytosis, cellular
migration, and chemotaxis (14-17). In vascular smooth muscle cells IAP
also associates with the 21 integrin and
modulates the chemotactic response of these cells to soluble collagen
(18). Numerous TM4SF members have been shown to associate with
1 integrins and with other TM4SF members (19-27). For
example, the TM4SF molecule CD63 has been shown to specifically
associate with 31 and
61 integrins (21), and the
CD63-31 complex was found to be
associated with phosphatidylinositol 4-kinase activity, suggesting a
novel integrin-signaling pathway (28). Furthermore, transfection
studies with CD63 in a melanoma cell line resulted in marked inhibition of cell motility, an effect probably mediated by its influence on
1 integrin function (29). Similarly, another TM4SF
member, CD9, also has been shown to associate with 1
integrins and to be implicated in cellular signaling, adhesion,
motility, and differentiation (19, 20, 22, 30-32). It is believed that
both IAP and the TM4SF molecules complex with integrins via
extracellular domains, since the Ig domain of IAP is essential for
effects on v3 integrin function (33) and
the 1 integrin association with CD63 occurs independently of the integrin -subunit cytoplasmic tail (20). More
recently, Yaunch et al. (34) demonstrated using chimeras that the CD151 (TM4SF)/31 association
required extracellular domains found in CD151 and the -chain of the
integrin. Furthermore, in K562 cells, in which the
47 integrin initiates cell adhesion under
flow conditions, the extracellular domains of the integrin determine
its localization to microvilli, where both IAP and TM4SF proteins were
also found to be strongly concentrated (35). Given the ubiquitous
expression of both IAP and TM4SF, this suggests that regulation of
integrin localization and function by these accessory molecules may
play a widespread role in cellular processes.
1 integrin activation in a genetic
complementation assay (36), and a physical association between CD98 and
certain 1 integrins has recently been described (cited
in Ref. 36); in the case of CD98, however, the association with
1 integrins appears to occur between cytosolic domains
of the two glycoproteins. The CD98 interaction with 1
integrins appears to modulate the activation of T-cells (38) as well as
the adhesion of breast carcinoma cells (39). Other examples of
integrin-associated molecules that may modify or regulate integrin
function include the ErbB-2 oncogene, a receptor tyrosine kinase
associated with breast cancer progression in vivo. This
interaction demonstrated to facilitate phosphatidylinositol
3-kinase-dependent in vitro cell invasion by
erbB-2-transformed 3T3 cells (39). The cancer metastasis-promoting molecule, urokinase plasminogen activator receptor
(uPAR) also associates with and regulates 1 and
2 integrin function (40).
IIb3 integrin on the surface of platelets
(43), and although the functional consequence of this association is
not known, it has been shown that platelet activation achieved by the
addition of anti-CD36 antibodies involves signaling through this
integrin (44, 45). It is not known whether CD36 directly associates with integrins on other cell types (12), but there are several lines of
evidence to suggest that certain functions ascribed to CD36 directly
involve or implicate involvement of integrins.
v3
integrin, possibly with involvement of the extracellular matrix protein
thrombospondin functioning as a molecular bridge (46). Additionally,
both CD36 and the v5 integrin have been
shown to be involved in the phagocytosis of shed photoreceptor rod
outer segments by retinal pigment epithelial cells (47, 48).
Interestingly, CD36 functions as an adhesive cellular receptor for both
thrombospondin (49) and collagen (50), specificities that also overlap
with integrins of both the v and 1
integrin subfamilies (1, 11). Fluorescence-activated cell sorting of
C32 melanoma cells into populations expressing either high or low
levels of CD36 showed that high CD36 expression defined a more
fibroblastoid cell phenotype, with this population demonstrating
enhanced tumor growth and motility in in vivo studies using
nude mice (51). Additionally, transfection of CD36 into human umbilical
vein endothelial cells resulted in aberrant morphological changes
affecting in vitro tube formation (52). Taken together, these data suggest that certain functions attributed to CD36 directly involve or implicate involvement of integrins, particularly
phagocytosis, cell adhesion, and migration. How this accessory function
is enacted is not known.
1 integrins may physically and
functionally associate with caveolin. For example, caveolin-1 can
function to link Fyn kinase with the 1 integrin
subunit, this resulting in downstream signaling promoting cell cycling
(63). Furthermore the GPI-linked uPAR, 1
integrins and caveolin-1 co-immunoprecipitate in a single complex, and
this complex appeared necessary for integrin-mediated adhesion and
signaling (40, 64). Interestingly some Src-family kinases, including
Fyn, Lyn, and Yes known to associate with CD36 (65, 66) are also
associated with DIGs (56, 57, 67), suggesting that CD36 signaling and
perhaps other CD36-mediated functions may be enacted via raft domains.
31 and
61, and this association was also shown
on C32 melanoma cells expressing endogenous CD36. We then used
transient transfection of chimeric constructs of CD36 to demonstrate
that the association between CD36 and 1 integrins requires the extracellular domain of the CD36 molecule. Finally, this
association may occur within raft domains/DIGs, since ectopic expression of CD36 increased the proportion of 1
integrin(s) found within this fraction.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and 5 integrin subunits, respectively,
and polyclonal (pAb) anti-1 integrin (69) were generous
gifts of Dr. Ken Yamada (National Institutes of Health, Bethesda, MD).
mAbs 13C2 and 23C6, directed against the v integrin
subunit and the v3 complex, respectively,
were gifts from Dr. M. Horton (Imperial Cancer Research Fund,
St. Bartholomew's Hospital, London). AK7 (anti-2
integrin), VM58 (anti-CD36) mAbs, and affinity-purified polyclonal
anti-CD36 (70) were gifts from Dr. M. C. Berndt (Baker Institute,
Victoria, Australia). IH4 anti-ICAM-1 and IA7 and IE8 anti-CD36 mAbs
were provided by Dr. A. W. Boyd (Queensland Institute of Medical
Research, Australia). E7P6 against 6 integrin was
provided by Dr. M. V. Agrez (University of Newcastle, Australia).
Polyclonal anti-calreticulin (LAR-090) and FMC56 (anti-CD9 mAb) were
gifts of Dr. S. Dedhar (University of British Columbia, Canada) and Dr.
H. Zola (Flinders Medical Center, Adelaide, Australia), respectively.
mAb 11H5 directed against CD36 was provided by Dr. P. Simmons (Hanson
Center, Adelaide). HB57 (anti-human µ chain), L230
(anti-v integrin), and AP3 (anti-3 integrin) were obtained from the ATCC. Mouse anti-1,
4, and 6 integrin subunits (TS2/7, 44H6,
and 4F10, respectively) and the anti-2 and
6 fluorescein isothiocyanate conjugates (AK7 and 4F10)
were purchased from Serotec Ltd. (Oxford, UK). Fluorescein isothiocyanate-conjugated anti-3 integrin was from
Southern Biotechnology Associates (Birmingham, AL). P1B5 (anti-3
integrin), M2 (anti-FLAG), and nonimmune mouse serum were purchased
from Life Technologies, Inc., Eastman Kodak Co., and Vet Services
(Adelaide, Australia), respectively. Polyclonal anti-caveolin was
purchased from Transduction Laboratories (Lexington, KY).
TM.CD36), the pEF.BOS.CD36
vector was used as template DNA for polymerase chain reaction
amplification using the oligonucleotides
5'-ggaggtattctaTCTAGAAtGggagacctgc-3' and 5'-ccccagggccatgcctcccagc-3'.
The amplification mutated valine 29 in CD36 to methionine (just outside
the predicted amino-terminal transmembrane) and introduced an
XbaI restriction site. The polymerase chain reaction product
was then restricted with XbaI and subcloned to the
XbaI site of the sIL3.FLAG.pEF.BOS vector, a derivative of
pEF.BOS containing the IL-3 signal sequence peptide upstream of a FLAG
epitope sequence (a gift of Dr. A. W. Boyd). The resulting construct encodes the IL-3 signal peptide, the FLAG epitope, and the
CD36 peptide beginning at glycine residue 30 of the wild-type CD36
sequence. All custom synthesis oligonucleotides were purchased from
Bresatec (Adelaide, Australia). As required, polymerase chain reaction
products were T/A-cloned (pGEM-T, Promega Corp., Madison, WI) before
restriction digestion. All vector constructs were validated by
automated sequencing analysis.
1 integrins, in some experiments
immunoprecipitations were weighted for CD36 detection by using 8-9
times the input cell lysate relative to the integrin
immunoprecipitations. The beads were then washed four times in lysis
buffer, eluted with Laemmli sample buffer, and resolved by SDS-PAGE.
Gels containing radioiodinated samples were fixed, stained, and dried,
and the labeled protein bands were visualized by autoradiography. Gels
containing biotinylated samples were transferred to nitrocellulose, and
the bands were visualized by blotting with streptavidin-biotin
horseradish peroxidase complexes (Amersham Pharmacia Biotech) as
described below for immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
31 and
61 Associate with CD36 on the Surface of
Melanoma Cells--
In order to determine whether CD36 influenced the
adhesion and migration of melanoma cells in a system amenable to
detailed analysis, we identified a well characterized metastatic
melanoma cell line (MV3; Ref. 68) that was deficient in CD36 expression and transfected this with CD36. Several bulk populations of
cells stably transfected with CD36 were derived by G418 selection, and one (uncloned) population expressing levels of CD36 comparable with
that endogenously expressed by C32 and WM115 melanoma cells was
selected for further study (MV3.CD36, Fig.
1A). In contrast to the report
of Wong et al. (51), who found that C32 melanoma cells
expressing high levels of CD36-antigen displayed a more fibroblastoid
appearance relative to low CD36-antigen-expressing cells, both control
(pEF.BOS vector) and CD36-transfected MV3-melanoma cells appeared
morphologically indistinguishable (Fig. 1B), and we could
detect no difference in the growth rates of the two populations (data
not shown). Adhesion assays measuring attachment to fibronectin, collagen, or laminin substrates showed no significant differences between the MV3.BOS and MV3.CD36 cells either measured under time- or
matrix protein concentration-dependent conditions (data not shown). In contrast, in cell migration assays carried out in Transwell chambers, the CD36-expressing cells were found to display significantly increased migration on fibronectin, laminin, and collagen substrates but not on the BSA substrate control (Fig. 1C). Wong
et al. (51) did not measure cell migration in
vitro, but the high CD36-expressing C32 cells were more metastatic
in vivo, and this, together with a more fibroblastoid
morphology, suggests that these cells would be more migratory than the
low CD36-antigen expressing cells. We did not test this directly by
cell sorting, but for the C32 mixed population the corresponding
migration rates were 32.4 ± 3.3, 13.7 ± 11.9, 28.7 ± 6.0, and 1.5 ± 0.7% on fibronectin, collagen, laminin, and BSA
(control) substrates, respectively. The MV3 cells are highly metastatic
in vivo (68) even in the absence of CD36 expression, and
in vitro the nontransfected parental cells already display a
somewhat motile appearance; therefore, it is perhaps not surprising
that CD36 expression did not alter the morphological appearance of
these cells. However, the significant increase in the already high
motility of these cells on extracellular matrix components indicates
that CD36 expression functionally interacts with the motility-promoting
machinery of these cells.
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Fig. 1.
Ectopic expression of CD36 in MV3 human
melanoma cells results in increased cell migration on extracellular
matrices. A, flow cytometric analysis of CD36
expression on human melanoma cell lines. C32 and WM115 cell lines,
which endogenously express CD36, and control and CD36 stably
transfected MV3 melanoma cell populations (MV3.BOS and MV3.CD36,
respectively) were analyzed by flow cytometry as described under
"Experimental Procedures." Open profiles
represent reactivity with the control mAb (HB57) in comparison with the
black profile (anti-CD36, VM58). B,
phase-contrast micrographs of the transfected MV3 melanoma cells shown
in A. C, in vitro haptotactic
migration assays of MV3.BOS and MV3.CD36 cells toward fibronectin
(FN), collagen type I (COL), laminin
(LM), and BSA. Significant differences were observed between
BOS and CD36 cells on the ECM substrates as shown (*, p < 0.05; **, p < 0.001 by paired Student's
t test).
1 integrins. As shown in Fig.
2A, the CD36 antibody immunoprecipitated a band at the position of CD36 (~85 kDa) together with two bands coincident with the immunoprecipitated 1
integrins (Fig. 2A). The associated protein bands were
absent from control immunoprecipitations (Fig. 2A) and from
CD36 immunoprecipitates of nontransfected MV3 cells (data not shown). A
weak band at ~85 kDa corresponding to the mobility of CD36 was also
observed in the 1 integrin precipitate. Due to the
relative abundance and labeling intensity of 1 integrins
and also because CD36 is relatively poorly immunoprecipitated from the
cell surface, an 8:1 lysate input weighting for the CD36
immunoprecipitation was used. This result appeared to suggest that CD36
was physically associating with 1 integrins in the
transfected MV3 cells.
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Fig. 2.
1 integrins
co-precipitate with CD36 in both transfected and endogenously
expressing melanoma cells. A, MV3.CD36 cells (~6 × 107 cells) were cell surface-labeled with radioiodine
and lysed in 1% Brij 96 lysis buffer, and the lysate was subjected to
immunoprecipitation analysis (i.p.) as described under
"Experimental Procedures." The lysate was divided such that 80% of
the material was used for specific immunoprecipitation for CD36 (VM58,
IA7, and IE8 mAb mixture) and 10% each for the control
immunoprecipitation (Cntrl; protein A beads alone) and
immunoprecipitation with the 1 pAb. Immunoprecipitates
were resolved by 7.5% SDS-PAGE under reducing conditions and
visualized by autoradiography after 2 days. B, WM115 and C32
melanoma cells (~6 × 107 cells) were analyzed as
described for A. Autoradiography was performed for 8 days.
1 integrins, although in these assays no CD36
was observed in the 1 integrin immunoprecipitate under
these conditions (Fig. 2B). These results suggest that the putative 1 integrin(s) association with CD36 is not
restricted to transfected cells but is representative of melanoma cell lines.
1 to
6 subunits known to associate with the 1
integrin subunit (1, 11). By this analysis, the 2,
4, and 6 integrin subunits appeared to be
most abundant with relatively lesser amounts of 1,
3, and 5 antigen (Fig.
3A). These cells have been
reported not to express v3 (73), and we
confirmed these results, although the cells were found to express
v5 (data not shown). In addition, MV3 cells are known not to
express 64 integrins (74).
Immunoprecipitation analysis of Brij 96 lysates from surface
radioiodinated MV3-CD36 cells substantiated these findings, although in
this case the 5 integrin in particular appeared to label
or precipitate less well or to be less abundant. In this experiment, it
was also noted that 3 and 6 integrin
subunit antibodies precipitated a labeled band with the same migration
as immunoprecipitated CD36 (Fig. 3B). This result suggested
that 3 and 6 are the predominant 1 integrins associating with CD36 in the transfected MV3
cells. To substantiate this notion, surface-labeled MV3-CD36 cells were lysed in Brij lysis buffer and CD36 immunoisolated with anti-CD36 antibodies directly coupled to Sepharose 4B beads. Bound proteins were
eluted from the beads with brief acid treatment and neutralized, and
aliquots of the eluate were reprecipitated with antibodies to the
1 and 1-6 integrin
subunits. The results (Fig. 3C) indicated that the major
integrin to co-precipitate with CD36 was
31 with trace amounts of
61 also associating with CD36. In repeat
experiments confirming these results, we found in addition by
reprecipitation with antibodies to v, 3,
5, and 6 that none of these
v integrins were associated with CD36 on the transfected
MV3 cells (data not shown).
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Fig. 3.
Integrins
31
and
61
specifically associate with CD36 in transfected MV3 and C32 melanoma
cells. A, the expression of the integrin
1, 2, 3, 4,
5, and 6 subunits on MV3 parental cells
was examined by flow cytometry. Open profiles
represent reactivity with the control mAb (HB57) in comparison with the
black profile (the indicated anti-integrin
-subunit mAbs). B, MV3.CD36 cells (~12 × 107 cells) were cell surface-labeled with radioiodine and
lysed in 1% Brij 96 lysis buffer, and the lysate was subjected to
immunoprecipitation analysis as described under "Experimental
Procedures." The lysate was divided equally, and specific
immunoprecipitations (i.p.) were performed using the
anti-CD36 specific mAbs (IA7 and IE8) directly coupled to Sepharose 4B
(lane 1) or with mAbs to the integrin subunits
1, 2, 3, 4,
5, and 6 (lanes
2-7, respectively). Immunoprecipitates were resolved on
7.5% SDS-polyacrylamide gels under nonreducing conditions and
visualized by autoradiography after 6 days. C, MV3.CD36
cells were radiolabeled as described for B, and the cell
lysate was immunoprecipitated using 500 µl of IA7/IE8-Sepharose
beads, before acid elution of the bound antigen(s) as described under
"Experimental Procedures." The eluate was divided equally, and
secondary immunoprecipitations were performed for CD36 (lane
1), the 1 integrin subunit (mAb 13;
lane 2), or the integrin subunits
1, 2, 3, 4,
5, and 6 (lanes
3-8, respectively). Immunoprecipitates were resolved on
7.5% SDS-PAGE gels under nonreducing conditions and visualized by
autoradiography after 6 weeks. D, MV3.CD36 cells were lysed
and immunoprecipitated as for B. The cell lysate (~25 µg
of protein; lane 1) and specific
immunoprecipitations for either CD36 (IA7/IE8; lane
2), and the integrin subunits 2,
3, and 6 (lanes
3-5, respectively) were resolved by SDS-PAGE under reducing
conditions. The gel was transferred to nitrocellulose, and
immunoblotting was performed with the CD36 pAb as described under
"Experimental Procedures." E, C32 melanoma cells
(~12 × 107 cells) were radiolabeled and lysed as
described for B. The lysate was first immunoprecipitated for
CD36, the bound antigen(s) was eluted, and then secondary
immunoprecipitation was performed as described in C for
either CD36 (lane 1) or the 1
integrin subunits 1, 2, 3,
4, 5, and 6
(lanes 2-7, respectively). Immunoprecipitates
were resolved on 7.5% SDS-PAGE gels under nonreducing conditions and
visualized by autoradiography after 12 weeks. For all experiments
shown, similar results were obtained on at least two separate
occasions.
2, 3,
and 6 integrins. When the resulting immunoprecipitates
were immunoblotted for CD36, both 3 and 6 lanes showed co-precipitated CD36, whereas 2 did not.
Taken together, these results suggested that
31 and 61
form a physical association with CD36 on the surface of transfected MV3
cells. To ensure that the association also applied to endogenous CD36
on the surface of melanoma cells, the elution and reprecipitation
experiment described above was repeated with C32 melanoma cells. These
cells also express each of the integrins
1-6, although 1,
2, 4, and 5 are relatively
less abundant by both flow cytometry and immunoprecipitation analyses
(data not shown). With the C32 melanoma cells, reprecipitation of the
integrins from the acid eluate obtained from the CD36 immunoprecipitate
revealed associated 31, but in this case
not 61 (Fig. 3E), but it is
likely that the relatively weaker association with
61 seen with the MV3 cells (Fig.
3C) would not be seen in precipitates from C32 cells in
which the precipitated signal is much weaker.
1 integrins
and CD36 occurred in vivo and to identify the cellular
location(s) of this association, dual color immunofluorescence and
confocal microscopic analysis were performed on the transfected MV3
cells after staining at the cell surface for 2 or
3 integrins and CD36. No specific association between
2 integrin and CD36 integrin was identified in these
cells (data not shown). In contrast, there was strong co-localization
between CD36 and 3 integrin in a high proportion of the
cells examined, and the sites of association were identified as being
predominantly on cell surface projections and/or membrane ruffles (Fig.
4).
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Fig. 4.
Co-localization of the
31
integrin with CD36 in transfected MV3 melanoma cells by confocal
microscopy. MV3.CD36 cells adhered to glass coverslips were
immunostained for both the 3 integrin subunit and CD36
as described under "Experimental Procedures." Two captured
images are shown: 3 (green) and CD36 (red)
channel images are merged to show areas of co-localization (highlighted
by arrowheads). Bar, 10 µm.
1
integrin association, chimeras between CD36 and ICAM-1 were utilized
(Fig. 5A). These constructs
were transiently transfected into the MV3 melanoma cells, and 3 days
following transfection, the cells were surface-labeled with biotin,
lysed in Brij 96 lysis buffer, and immunoprecipitated with antibodies
directed against CD36, ICAM-1, and 1 integrins. The
results demonstrate that, as was shown for the permanently transfected
MV3.CD36 cells, immunoprecipitates of CD36 from MV3 cells transiently
transfected with wild-type CD36 co-precipitated 1
integrins (Fig. 5B); note that using the biotinylation
protocol, the -subunit(s) of 1 integrins are less
strongly labeled in comparison with radioiodination (compare with Fig.
3B). The substitution of the CD36 carboxyl-terminal
cytoplasmic tail with that of ICAM-1 did not affect the
1 integrin association in the transfected cells (Fig.
5B; CD36/ICAM tail). Additionally, as demonstrated for
wild-type CD36 above (Fig. 3D), immunoblotting of the
immunoprecipitates of specific 1 integrins with
antibodies against CD36 revealed that the CD36/ICAM tail chimera
co-precipitated with 3 and 6 integrins
but not with the 2 subunit (Fig. 3C),
corroborating the results presented in Fig. 5B and further
confirming the specificity of the CD36-1 integrin
interaction.
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Fig. 5.
The association with
1 integrins does not involve the
carboxyl-terminal transmembrane/cytoplasmic domains of CD36.
A, schematic representation of CD36, ICAM-1, and chimeric
constructs. The hatched areas represent sequences
derived from ICAM-1. TM indicates the location of the
predicted transmembrane domains. The wild-type CD36, wild-type ICAM-1,
CD36/ICAM tail, and ICAM/CD36 constructs encode 472, 532, 497, and 492 amino acids, respectively. B, MV3 cells (~2 × 107 cells) were transiently transfected with the empty
pEF.BOS vector (BOS), wild-type CD36, or the CD36/ICAM tail
chimera (CITail). Following transfection, the cells were
biotin-labeled on the cell surface and lysed in 1% Brij lysis buffer,
and immunoprecipitations (i.p.) were performed for CD36
(VM58, IA7, and IE8 mAb mixture) using 90% of the total cell lysate
and for 1 integrins (1 pAb) using
10% of cell lysate. The samples were resolved by 7.5% SDS-PAGE under
reducing conditions and transfered to nitrocellulose, and biotinylated
proteins were visualized with streptavidin-horseradish peroxidase as
described under "Experimental Procedures." C, MV3 cells
(~10 × 107 cells) were transiently transfected with
the CD36/ICAM tail construct before lysis with 1% Brij lysis buffer.
Immunoprecipitations were performed for CD36 (11H5 mAb) and for the
indicated integrin subunits 2, 3, and
6 before resolving the samples by SDS-PAGE under
reducing conditions. The gel was transferred to nitrocellulose, and
immunoblotting was performed for CD36 as described under
"Experimental Procedures." D, MV3 cells (~2 × 107 cells) were transiently transfected with either CD36,
BOS, or the ICAM/CD36 (I/CD36) chimera and analyzed as
described for B. The lysates were subjected to
immunoprecipitation with either the anti-CD36 mAb mixture (CD36;
IA7/IE8), or with anti-ICAM-1 (BOS and I/CD36; IH4 mAb). Note that the
increased migration seen with the expressed ICAM/CD36 construct results
from differences in glycosylation. All results were confirmed in three
separate experiments.
1 integrins and CD36 occurs in the absence of the
carboxyl-terminal cytoplasmic tail of CD36. However, the predicted CD36
polypeptide contains 471 amino acids with hydrophobic regions
resembling transmembrane domains present adjacent to both the amino-
and carboxyl-terminal regions. The short amino-terminal hydrophobic
region resembles an uncleaved signal peptide (75) with only the
initiator methionine being absent in the mature protein (76). From
these data, it has been proposed that CD36 is ditopic in an "inverted
horseshoe" membrane configuration (41), although it has been
controversial as to whether the amino-terminal transmembrane domain
actually serves to anchor the protein (77-79). If the amino-terminal
regions does form a transmembrane domain, therefore, it is possible
that an association between the 1 integrins and CD36
could occur in this region, either in the amino-terminal cytoplasmic
domain or in the transmembrane domain.
TM.CD36; Fig. 6A). Upon
transfection, however, this construct was very poorly expressed in the
MV3 cells. Preliminary analyses revealed that reasonable levels of
expression could be obtained with this construct in transiently
transfected COS-7 cells (data not shown). Therefore, COS-7 cells were
transfected with the control BOS vector, wild-type CD36, or the
FNTM.CD36 construct, and the lysates of biotin-labeled cells were
subjected to immunoprecipitation analysis. Immunoprecipitates of
wild-type CD36 showed a specific co-precipitated band with identical
mobility to the 1 integrin subunit (Fig. 6B),
indicating that the transfected human CD36 can associate with
endogenous monkey 1 integrin(s). Similarly, both the
amino-terminal truncate of CD36 (FNTM.CD36; Fig. 6B) and
the CD36/ICAM tail mutant (Fig. 5A and data not shown) also co-precipitated endogenous 1 integrins from these cells.
Taken together, these results establish that the association between 1 integrins and CD36 requires the extracellular domain
of CD36.
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Fig. 6.
The extracellular domain of CD36 is
implicated in its association with
1 integrins. A,
schematic representation of wild-type CD36 and the epitope-tagged
amino-terminal truncation of CD36 (FNTM.CD36). TM,
IL3, and F indicate the locations of the
predicted transmembrane domains, the IL3 signal sequence, and the FLAG
epitope tag sequence, respectively. B, COS-7 (~2 × 107 cells) were transiently transfected with either pEF.BOS
(control), CD36, or the FNTM.CD36 construct and analyzed as
described in the legend to Fig. 5. The lysates were subjected to
immunoprecipitation (I.P.) either with the anti-CD36 mAb
mixture (BOS and CD36; VM58/IA7/IE8 mAbs) or with anti-FLAG (FNTM;
M2 mAb). Ten percent of the total lysate for each transfection was
immunoprecipitated for 1 (pAb). The strong band denoted
by the asterisk represents a nonspecific band precipitated
with the M2 mAb. Results were confirmed in two separate
experiments.
1 Integrin
to DIGs in Transfected MV3 Melanoma Cells--
Membrane
microdomains, or rafts, are specialized compartments of the plasma
membrane that are highly enriched in cholesterol and glycosphingolipids
(53-55, 80). They are also greatly enriched in many signal-transducing
molecules and are therefore thought to represent signaling microdomains
within the membrane (56, 57, 67). These rafts are relatively insoluble
in detergents such as Triton X-100 by virtue of their lipid
constituents, and this feature has enabled the well validated
biochemical approach of DIG isolation to identify the proteins located
within the microdomains (53, 56, 67, 80). Relatively few transmembrane
glycoproteins have been identified as raft-associated in this way.
However, CD36 has been characterized as one such molecule that has been shown to partition into DIGs isolated from murine lung endothelial cells (56), human platelets (57), and transfected COS-7 cells (58),
where it may engage in signal transduction (44, 65, 81). We therefore
determined whether CD36 also partitioned into DIGs isolated from the
transfected MV3 cells, and, because this may provide a possible
mechanism for the influence of CD36 on integrin function, we examined
whether CD36 expression altered the proportion of 1
integrin contained in DIGs.
1
integrin subunit was observed predominantly in the soluble region of
the gradient from both CD36 and control transfectants, the upper band
representing the mature molecule and the lower band corresponding to
the precursor (69). However, whereas in the control (MV3.BOS)
transfectants a small amount of 1 integrin was found in
DIGs, being entirely the mature form, the proportion of this mature
protein found in the DIG fraction was considerably relatively enriched
in fractions from the CD36 transfectants. CD9, a member of the TM4SF
family and known to form a physical association with 1
integrins (20, 22, 23, 25, 32), was also examined for its distribution in these gradients. This molecule was well represented in most fractions but was most enriched in fractions 5 and 6 just below the
DIGs and above the "soluble" region of the gradient. These fractions do not contain much biotinylated material from lysates of
cells surface-labeled with biotin, and they are also enriched in the
Golgi marker GS282;
therefore, they may represent intracellular material. Notably, however,
there appeared to be no difference in the relative amounts of CD9 in
the DIG fractions from the control- or CD36-transfected cells.
Densitometric analyses of the data confirmed these observations.
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Fig. 7.
Biochemical analysis of sucrose gradient
fractions isolated from control- and CD36-transfected MV3 melanoma
cells. MV3.BOS cells (A) or MV3.CD36 cells
(B) (~2 × 107 cells) were lysed with 1%
Triton X-100 in MES-buffered saline (pH 6.5) and subjected to sucrose
density gradient fractionation, SDS-PAGE, and immunoblotting with the
indicated antibodies. The immunoblots represent equal protein loading
(5 µg of protein/fraction) of the 12 × 1-ml fractions collected
from the top of the sucrose gradient. The boxed
fractions indicate the location of the light-refractive
DIGs. Immunoblots with polyclonal antibodies against CD36, caveolin,
and calreticulin were assayed under reducing conditions, whereas
polyclonal anti- 1 integrin and anti-CD9 (mAb FMC56) were
analyzed under nonreducing conditions of electrophoresis (7.5-12%
gradient SDS-PAGE). C, enrichment of each antigen in DIGs
(determined from the immunoblots shown in A and
B) was determined by densitometric analysis as described
under "Experimental Procedures." Similar distributions were found
for each antigen in at least two separate experiments.
1 integrin found in the DIG fraction, although
small, was found to be increased ~4-fold in the CD36 transfectants
compared with the vector-only control-transfected cells (17.8 versus 4.5%, respectively). CD9 was distributed across the
gradient, with a substantial proportion found in the insoluble
fractions; however, this distribution was unaltered between the two
transfected cell populations.
1 integrin into membrane microdomains, where it is more
likely to encounter signaling molecules that could modulate the
function of the integrins. It is possible that substantially higher
proportions of 31 and
61 were specifically sequestered, but
blotting antibodies to the -subunits were not available for this study.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 integrins
31 and 61,
and this association requires the extracellular domain of CD36;
co-expression of CD36 in MV3 melanoma cells results in increased
migration on extracellular matrix components, indicating that the
association has functional consequences. This requirement for the
extracellular domain of CD36 for integrin association is shared with
other molecules laterally associated with integrins, including
IAP, the GPI-linked uPAR, and, notably, members of the TM4SF
family (20, 24, 33-35, 40).
31 and, to a lesser extent,
61. It is striking that these particular
integrins are also those most frequently identified as associating with
members of the TM4SF family (21, 22, 25, 27, 28, 32). In order to
demonstrate the association of CD36 and integrins by
co-immunoprecipitation, we lysed the cells in Brij 96 detergent. Some
co-precipitation (although less) was also demonstrated after cell lysis
in 1% octyl glucoside, but the association was disrupted after lysis
of the cells in 1% Nonidet P-40 or 1% Triton X-100.2
These features are shared with several reported TM4SF-integrin associations, and Hemler and associates (21, 22, 24, 25) have
rigorously demonstrated that such associations are not the result of
detergent artifact but rather depend upon the strength of the
association and its capacity to withstand "stringent" detergents. Some suggested TM4SF-integrin associations have been based upon co-immunoprecipitation results after lysis in CHAPS, Brij 99, or Brij
58 but do not withstand lysis in Brij 96 (as used in the present
study), which is more hydrophobic; therefore, the CD36-integrin association is reasonably robust. Recently, however, Yaunch et al. (26, 34) reported a direct association between an
extracellular domain of the TM4SF protein CD151 and integrin
31 that occurred at high stoichiometry
even after cell lysis in 1% Triton X-100 and was relatively resistant
to denaturing detergents. Further, an association between CD151 and
31 integrin was found on every cell and
tissue type examined. CD151 can also associate with other TM4SF
proteins under milder conditions of lysis (26), leading these authors
to postulate that some accounts of TM4SF-integrin association may be
due to co-precipitation with CD151-31
complexes. These findings suggest the possibility that CD36 may form
part of a ternary complex with the integrin and CD151. Perhaps in
support of this, in a chemical cross-linking study of platelets, Dorahy et al. (43) demonstrated that the platelet integrin
IIb3 formed a physical association with
both CD36 and the TM4SF protein, CD9. Studies are in progress to
determine whether CD36 associates with CD151 in melanoma cells, but it
should be noted that whereas CD151 associates with the immature
3 precursor (34), CD36 was found to associate only with
the mature 1 molecule.
1
integrin contained within the DIG fraction isolated from MV3 melanoma cells transfected with CD36 was relatively enriched compared with control cells. Several studies have validated the concept that such
biochemically isolated DIGs are representative of specialized plasma
membrane microdomains or rafts (53, 56, 67, 80). Caveolin, the marker
coat protein of the distinct membrane structures known as caveolae was
also found in this fraction, raising the possibility that association
with CD36 facilitates the entry of 1 integrins into
caveolae. Caveolae are thought to bud from membrane microdomains in a
process controlled by caveolin oligomers, and, in addition to their
characteristic enrichment in cholesterol and sphingolipid, they are
greatly enriched in cellular signaling proteins (56, 57, 67). Integrins
are not generally considered to locate to caveolae (56, 62), but recent
work has revealed that a fraction of 1 integrin in
Fisher rat thyroid cells associates with both caveolin and the
protein-tyrosine kinase Fyn, and Fyn-dependent Shc-mediated
signaling in response to integrin ligation was found to be dependent
upon the co-expression of caveolin-1 (63). Also, by use of antisense
methodology in kidney 293 cells, Wei et al. (64) found that
caveolin was important to 1
integrin-dependent adhesion to fibronectin and the
activation of focal adhesion kinase. The authors propose that
1 integrins form a complex with caveolin that is
stabilized by the nonintegrin, GPI-linked urokinase receptor, uPAR;
this complex, in turn, regulates the ability of caveolin to regulate
members of the Src family of protein-tyrosine kinases surrounding integrins.
1 integrins through its
external domain, CD36 can substitute for or mimic uPAR in providing a
bridge for caveolin association. Three observations militate against
this possibility. First, in our co-precipitation studies, we were
unable to detect any caveolin in the CD36/1 precipitates by
immunoblotting.2 Second, in an immunoultrastructural study
of the CD36-transfected MV3 cells, it was found that the CD36 appeared
to compete with caveolin such that cells expressing high levels of CD36
displayed considerably fewer of the flask-like invaginations
representative of morphological
caveolae.3 The reason for
this is not known at present, but this result does suggest that CD36 is
not simply a passive facilitator of caveolae association. Third, CD36
locates to biochemically isolated plasma membrane rafts even in the
absence of caveolin; in platelets that lack caveolin expression, CD36
is found highly enriched in the DIG fraction together with active Lyn
protein-tyrosine kinase (57, 81). Further, in these enucleate cells,
CD36 physically associates with the platelet integrin
IIb3 (43) and with the tyrosine-protein
kinases, Fyn, Lyn, and Yes (65). CD36 itself may bind cholesterol (86)
and fatty acids (87) and co-precipitates with Src family
protein-tyrosine kinases from a number of other cell types, including
melanoma cells (65, 66). Together, these data suggest that CD36 may
play a role in guiding integrins to signaling rafts. Many hemic cells
lack caveolin expression and do not form caveolae (88), and the
down-regulation of caveolin expression is a feature of cell
transformation (89). In contrast, CD36 expression is highly prominent
in hemic cells such as platelets and macrophages (41, 42) as it is on
melanoma cells in situ (90) and breast cancer cells (91). We
tentatively suggest, therefore, that in those cells expressing CD36 but
lacking caveolin expression, CD36 may play an important role in the
regulation of integrin function.
51
integrin to fibronectin or vitronectin (40), and the 67-kDa monomeric
laminin receptor that regulates the laminin-binding function of the
64 integrin (95). CD36 has been described as a cell surface receptor for both collagen and thrombospondin (49,
50). Tandon and colleagues (96, 97) have recorded that CD36 expression
influences the rate of platelet attachment and spreading on type I
collagen mediated by the 21 integrin. 31 integrin also can bind collagen;
however, in our study with transfected MV3 melanoma cells we could
detect no influence of CD36 expression on the degree or rate of
spreading of these cells on collagen or on the
61 substrate, laminin (data not shown). CD36 binding to the pericellular matrix glycoprotein, thrombospondin, may be a more attractive candidate for the modulatory role of CD36 in
cell migration. Thrombospondin is abundantly secreted by many migratory
cell types, including melanoma cells; it can bind to several
extracellular matrix proteins and, functioning as an antiadhesive
molecule, can promote cell migration (reviewed in Ref. 98).
Thrombospondin contains several distinct domains able to bind to
discrete cell surface receptors, including CD36 and v
and 1 integrins (98), and fibroblast migration on
thrombospondin has been shown to include a role for both integrins and
CD36 (99). Of interest, the integrin-associated protein (IAP or CD47),
which has been demonstrated to physically associate with integrins and to modulate their function (13-16, 33), also functions as a receptor for the carboxyl-terminal region of thrombospondin (17, 100). In a
recent study, Wang and Frazier (18) demonstrated that IAP physically
associated with 21 integrin on aortic
smooth muscle cells and that thrombospondin peptides able to bind IAP
stimulated the chemotaxis of those cells on gelatin-coated filters.
v3 integrin
because of reports of the coordination of these receptors in the
phagocytic uptake of apoptotic cells (46). However, we were unable to
demonstrate co-precipitation of these molecules from WM115 melanoma
cells that express both in abundance, or from MV3 cells transfected with both CD36 and 3.2 The ready
demonstration of a CD36-31 association
under these conditions suggests that, if CD36 and
v3 act coordinately in phagocytosis, they
do so without being in physical association. However, while a role for
CD36 in this function has been well validated in transfection studies
(101) and a CD36 family member, croquemort, has been found
in genetic studies to be essential for the removal of apoptotic cells
during Drosophila development (102), similar studies have
not been carried out to determine the role of
v3. Studies documenting a role for
v3 in this function have depended upon
antibody-induced or peptide agonist-induced inhibition (46, 103-106).
More recent work has established that the engagement of
3 integrins with antibody or ligand can result in the
down-regulation of the function of 1 integrins in a
signal-mediated process known as transdominant inhibition (8, 9).
Further, it has been established that 31
participates in phagocytosis mediated by human breast cancer cells
(107), most of which express CD36 (91). Therefore, the findings
reported here of an association between CD36 and
31 integrin may indicate that the
conclusions of studies implicating v3 in
the phagocytic process may warrant further evaluation to assess the
role of 1 integrins and to consider the possibility that
v3 engages in integrin receptor
cross-talk.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Hynes, R. O.
(1992)
Cell
69,
11-25
2.
Juliano, R. L.,
and Haskill, S.
(1993)
J. Cell Biol.
120,
577-585
3.
Clark, E. A.,
and Brugge, J. S.
(1995)
Science
268,
233-239
4.
Schwartz, M. A.,
Schaller, M. D.,
and Ginsberg, M. H.
(1995)
Annu. Rev. Cell Dev. Biol.
11,
549-599
5.
Giancotti, F. G.,
and Ruoslahti, E.
(1999)
Science
285,
1028-1032
6.
Monier-Gavelle, F.,
and Duband, J.-L.
(1997)
J. Cell Biol.
137,
1663-1681
7.
Rainger, G. E.,
Buckley, C.,
Simmons, D. L.,
and Nash, G. B.
(1997)
Curr. Biol.
7,
316-325
8.
Blystone, S. D.,
Graham, I. L.,
Lindberg, F. P.,
and Brown, E. J.
(1994)
J. Cell Biol.
127,
1129-1137
9.
Diaz-Gonzalez, F.,
Forsyth, J.,
Steiner, B.,
and Ginsberg, M. H.
(1996)
Mol. Biol. Cell
7,
1939-1951
10.
Porter, J. C.,
and Hogg, N.
(1997)
J. Cell Biol.
6,
1437-1447
11.
Dedhar, S.
(1995)
Cancer Metastasis Rev.
14,
165-172
12.
Hemler, M. E.
(1998)
Curr. Opin. Cell Biol.
10,
578-585
13.
Brown, E. J.,
Hooper, L.,
Ho, T.,
and Gresham, H. D.
(1990)
J. Cell Biol.
111,
2785-2794
14.
Zhou, M.-J.,
and Brown, E. J.
(1993)
J. Exp. Med.
178,
1165-1174
15.
Blystone, S. D.,
Lindberg, F. P.,
LaFlamme, S. E.,
and Brown, E. J.
(1995)
J. Cell Biol.
130,
745-754
16.
Cooper, D.,
Lindberg, F. P.,
Gamble, J. R.,
Brown, E. J.,
and Vadas, M. A.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
3978-3982
17.
Gao, A.-G.,
Lindberg, F. P.,
Finn, M. B.,
Blystone, S. D.,
Brown, E. J.,
and Frazier, W. A.
(1996)
J. Biol. Chem.
271,
21-24
18.
Wang, X. Q.,
and Frazier, W. A.
(1998)
Mol. Biol. Cell
9,
865-874
19.
Slupsky, J. R.,
Seehafer, J. G.,
Tang, S. C.,
Masellis-Smith, A.,
and Shaw, A. R.
(1989)
J. Biol. Chem.
264,
12289-12293
20.
Rubenstein, E.,
Le Naour, F.,
Billard, M.,
Prenant, M.,
and Boucheix, C.
(1994)
Eur. J. Immunol.
24,
3005-3013
21.
Berditchevski, F.,
Bazzoni, G.,
and Hemler, M. E.
(1995)
J. Biol. Chem.
270,
17784-17790
22.
Berditchevski, F.,
Zutter, M. M.,
and Hemler, M. E.
(1996)
Mol. Biol. Cell
7,
193-207
23.
Radford, K. J.,
Thorne, R. F.,
and Hersey, P.
(1996)
Biochem. Biophys. Res. Commun.
222,
13-18
24.
Mannion, B. A.,
Berditchevski, F.,
Kraeft, S.-K,
Chen, L. B.,
and Hemler, M. E.
(1996)
J. Immunol.
157,
2039-2047
25.
Tachibana, I.,
Bodorova, J.,
Berditchevski, F.,
Zutter, M. M.,
and Hemler, M. E.
(1997)
J. Biol. Chem.
272,
29181-29189
26.
Yauch, R. L.,
Berditchevski, F.,
Harler, M. B.,
Reichner, J.,
and Hemler, M. E.
(1998)
Mol. Biol. Cell
9,
2751-2765
27.
Yanez-Mo, M.,
Alfranca, A.,
Cabanas, C.,
Marazuela, M.,
Tejedor, R.,
Ursa, M. A.,
Ashman, L. K.,
de Landazuri, M. O.,
and Sanchez-Madrid, F.
(1998)
J. Cell Biol.
141,
791-804
28.
Berditchevski, F.,
Tolias, K. F.,
Wong, K.,
Carpenter, C. L.,
and Hemler, M. E.
(1997)
J. Biol. Chem.
272,
2595-2598
29.
Radford, K. R.,
Thorne, R. F.,
and Hersey, P.
(1997)
J. Immunol.
158,
3353-3358
30.
Ikeyama, S.,
Koyama, M.,
Yamaoko, M.,
Sasada, R.,
and Miyake, M.
(1993)
J. Exp. Med.
177,
1231-1237
31.
Shaw, A. R. E.,
Domanska, A.,
Mak, A.,
Gilchrist, A.,
Dobler, K.,
Visser, L.,
Poppema, S.,
Fliegel, L.,
Letarte, M.,
and Willett, B. J.
(1995)
J. Biol. Chem.
270,
24092-24099
32.
Jones, P. H.,
Bishop, L. A.,
and Watt, F. M.
(1996)
Cell Adhesion Commun.
4,
297-305
33.
Lindberg, F. P.,
Gresham, H. D.,
Reinhold, M. I.,
and Brown, E. J.
(1996)
J. Cell Biol.
134,
1313-1322
34.
Yauch, R. L.,
Kazarov, A. R.,
Desai, B.,
Lee, R. T.,
and Hemler, M. E.
(2000)
J. Biol. Chem.
275,
9230-9238
35.
Abitorabi, M. A.,
Pachynski, R. K.,
Ferrando, R. E.,
Tidswell, M.,
and Erle, D. J.
(1997)
J. Cell Biol.
139,
563-571
36.
Fenczik, C. A.,
Sethi, T.,
Ramos, J. W.,
Hughes, P. E.,
and Ginsberg, M. H.
(1997)
Nature
390,
81-85
37.
Warren, A. P.,
Patel, K.,
Miyamoto, Y.,
Wygant, J. N.,
Woodside, D. G.,
and McIntyre, B. W.
(2000)
Immunology
99,
62-68
38.
Chandrasekaran, S.,
Guo, N. H.,
Rodrigues, R. G.,
Kaiser, J.,
and Roberts, D. D.
(1999)
J. Biol. Chem.
274,
11408-11416
39.
Gambaletta, D.,
Marchetti, A.,
Benedetti, L.,
Mercurio, A. M.,
Sacchi, A,
and Falcioni, R.
(2000)
J. Biol. Chem.
275,
10604-10610
40.
Wei, Y.,
Lukashev, M,
Simon, D. I.,
Bodary, S. C.,
Rosenberg, S.,
Doyle, M. V.,
and Chapman, H. A.
(1996)
Science
273,
1551-1555
41.
Greenwalt, D. E.,
Lipsky, R. H.,
Ockenhouse, C. F.,
Ikeda, H.,
Tandon, N. N.,
and Jamieson, G. A.
(1992)
Blood
80,
1105-1115
42.
Thorne, R. F.,
Dorahy, D. J.,
Wilkinson, R. M.,
and Burns, G. F.
(2000)
in
Thrombosis and the Vessel Wall
(Berndt, M., ed)
, pp. 149-172, Harwood Academic Publishers, Chur, Switzerland
43.
Dorahy, D. J.,
Berndt, M. C.,
Shafren, D. R.,
and Burns, G. F.
(1996)
Biochem. Biophys. Res. Commun.
218,
575-581
44.
Aiken, M. L.,
Ginsberg, M. H.,
Byers-Ward, V.,
and Plow, E. F.
(1990)
Blood
76,
2501-2509
45.
Mazurov, A. V.,
Vinogradov, D. V.,
Vlasik, T. N.,
Burns, G. F.,
and Berndt, M. C.
(1992)
Platelets
3,
181-188
46.
Savill, J.,
Nancy, H.,
Ren, Y.,
and Haslett, C.
(1992)
J. Clin. Invest.
90,
1513-1522
47.
Ryeom, S.,
Sparrow, J.,
and Silverstein, R. L.
(1996)
J. Cell Sci.
109,
387-395
48.
Finnemann, S. C.,
Bonilha, V. L.,
Marmorstein, A. D.,
and Rodriguez-Boulan, E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12932-12937
49.
Asch, A. S.,
Barnwell, J.,
Silverstein, R. L.,
and Nachman, R. L.
(1987)
J. Clin. Invest.
79,
1054-1061
50.
Tandon, N. N.,
Kralisz, U.,
and Jamieson, G. A.
(1989)
J. Biol. Chem.
264,
7576-7583
51.
Wong, J. E. L.,
Asch, A. S.,
Silverstein, R. L.,
and Nachman, R. L.
(1989)
Blood
74 Suppl. 1,
638 (abstr.)
52.
Dawson, D. W.,
Pearce, F. A.,
Zhong, R.,
Silverstein, R. L.,
Frazier, W. A.,
and Bouck, N. P.
(1997)
J. Cell Biol.
138,
707-717
53.
Brown, D. A.,
and London, E.
(1997)
Biochem. Biophys. Res. Commun.
240,
1-7
54.
Simons, K.,
and Ikonen, E.
(1997)
Nature
387,
569-572
55.
Kurzchalia, T. V.,
and Parton, R. G.
(1999)
Curr. Opin. Cell Biol.
11,
424-431
56.
Lisanti, M. P.,
Scherer, P. E.,
Vidugiriene, J.,
Tang, Z.,
Hermanowski-Vosatka, A.,
Tu, Y. H.,
Cook, R. F.,
and Sargiacomo, M.
(1994)
J. Cell Biol.
126,
11-26
57.
Dorahy, D. J.,
Lincz, L. F.,
Meldrum, C. J.,
and Burns, G. F.
(1996)
Biochem. J.
319,
67-72
58.
Thorne, R. F.,
Meldrum, C. J.,
Harris, S. J.,
Dorahy, D. J.,
Shafren, D. R.,
Berndt, M. C.,
Burns, G. F.,
and Gibson, P. G.
(1997)
Biochem. Biophys. Res. Commun.
240,
812-818
59.
Ko, Y. G.,
Lee, J. S.,
Kang, Y. S.,
Ahn, J. H.,
and Seo, J. S.
(1999)
J. Immunol.
162,
7217-7223
60.
Manes, S.,
Mira, E.,
Gomez-Mouton, C.,
Lacalle, R. A.,
Keller, P.,
Labrador, J. P.,
and Martinez, A. C.
(1999)
EMBO J.
18,
6211-6220
61.
Rothberg, K. G.,
Heuser, J. E.,
Donzell, W. C.,
Ying, Y. S.,
Glenney, J. R.,
and Anderson, R. G.
(1992)
Cell
68,
673-682
62.
Mineo, C.,
James, G. L.,
Smart, E. J.,
and Anderson, R. G
(1996)
J. Biol. Chem.
271,
11930-11935
63.
Wary, K. K.,
Mariotti, A.,
Zurzolo, C.,
and Giancotti, F. G.
(1998)
Cell
94,
625-634
64.
Wei, Y.,
Yang, X.,
Liu, Q.,
Wilkins, J. A.,
and Chapman, H. A.
(1999)
J. Cell Biol.
144,
1285-1294
65.
Huang, M.-M.,
Bolen, J. B.,
Barnwell, J. W.,
Shattil, S. J.,
and Brugge, J. S.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
7844-7849
66.
Bull, H. A.,
Brickell, P. M.,
and Dowd, P. M.
(1994)
FEBS Lett.
351,
41-44
67.
Sargiacomo, M.,
Sudol, M.,
Tang, Z.,
and Lisanti, M. P.
(1993)
J. Cell Biol.
122,
789-807
68.
van Muijen, G. N. P.,
Jansen, K. F. J.,
Cornelissen, M. H. A.,
Smeets, D. F. C. M.,
Beck, J. L. M.,
and Ruiter, D. J.
(1991)
Int. J. Cancer
48,
85-91
69.
Akiyama, S. K.,
Yamada, S. S.,
Chen, W.-T.,
and Yamada, K. M.
(1989)
J. Cell Biol.
109,
863-875
70.
Berger, G.,
Caen, J. P.,
Berndt, M. C.,
and Cramer, E. M.
(1993)
Blood
82,
3034-3044
71.
Mizushima, S.,
and Nagata, S.
(1990)
Nucleic Acids Res.
18,
5322
72.
Burns, G. F.,
Lucas, C. M.,
Krissansen, G. W.,
Werkmeister, J. A.,
Scanlon, D. B.,
Simpson, R. J.,
and Vadas, M. A.
(1988)
J. Cell Biol.
107,
1225-1230
73.
Danen, E. H.,
Aota, S.,
van Kraats, A. A.,
Yamada, K. M.,
Ruiter, D. J.,
and van Muijen, G. N.
(1995)
J. Biol. Chem.
270,
21612-21618
74.
Danen, E. H.,
van Muijen, G. N.,
van de Wiel-van Kemenade, E.,
Jansen, K. F.,
Ruiter, D. J.,
and Figdor, C. G.
(1993)
Int. J. Cancer
54,
315-321
75.
Oquendo, P.,
Hundt, E.,
Lawler, J.,
and Seed, B.
(1989)
Cell
58,
95-101
76.
Tandon, N. N.,
Lipsky, R. H.,
Burgess, W. H.,
and Jamieson, G. A.
(1989)
J. Biol. Chem.
264,
7570-7575
77.
Asch, A. S.,
Liu, I.,
Bricetti, F. M.,
Barnwell, J. W.,
Kwakye-Berko, F.,
Dokun, A.,
Goldberger, J.,
and Pernambuco, M.
(1993)
Science
262,
1436-1440
78.
Pearce, S. F. A.,
Wu, J.,
and Silverstein, R. L.
(1994)
Blood
84,
384-389
79.
Tao, N.,
Wagner, S. J.,
and Lublin, D. M.
(1996)
J. Biol. Chem.
271,
22315-22320
80.
Brown, D. A.,
and Rose, J. K.
(1992)
Cell
68,
533-544
81.
Dorahy, D. J.,
and Burns, G. F.
(1998)
Biochem. J.
333,
373-379
82.
Michalak, M.,
Corbett, E. F.,
Mesaeli, N.,
Nakamura, K.,
and Opas, M.
(1999)
Biochem. J.
344,
281-292
83.
Wary, K. K.,
Mainiero, F.,
Isakoff, S. J,
Marcantonio, E. E.,
and Giancotti, F. G.
(1996)
Cell
87,
733-743
84.
Li, S.,
Okamoto, T.,
Chun, M.,
Sargiacomo, M.,
Casanova, J. E.,
Hansen, S. H.,
Nishimoto, I.,
and Lisanti, M. P.
(1995)
J. Biol. Chem.
270,
15693-15701
85.
Li, S.,
Couet, J.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
29182-29190
86.
Kronenberg, A.,
Grahl, H.,
and Kehrel, B.
(1998)
Thromb. Haemostasis
79,
1021-1024
87.
Abumrad, N. A,
el-Maghrabi, M. R.,
Amri, E. Z.,
Lopez, E.,
and Grimaldi, P. A.
(1993)
J. Biol. Chem.
268,
17665-17668
88.
Fra, A. M.,
Williamson, E.,
Simons, K.,
and Parton, R. G.
(1994)
J. Biol. Chem.
269,
30745-30748
89.
Koleske, A. J.,
Baltimore, D.,
and Lisanti, M. P.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
1381-1385
90.
Si, Z.,
and Hersey, P.
(1994)
Pathology
26,
6-15
91.
Greenwalt, D. E.,
Watt, K. W. K.,
So, O. Y.,
and Jiwani, N.
(1990)
Biochemistry
29,
7054-7059
92.
Chan, K. F.
(1988)
J. Biol. Chem.
263,
568-574
93.
Cheresh, D. A.,
Pierschbacher, M. D.,
Herzig, M. A.,
and Mujoo, K.
(1986)
J. Cell Biol.
102,
688-696
94.
Cheresh, D. A.,
Pytela, R.,
Pierschbacher, M. D.,
Klier, F. G.,
Ruoslahti, E.,
and Reisfeld, R. A.
(1987)
J. Cell Biol.
105,
1163-1173
95.
Ardini, E.,
Tagliabue, E.,
Magnifico, A.,
Buto, S.,
Castronovo, V.,
Colnaghi, M. I.,
and Menard, S.
(1997)
J. Biol. Chem.
272,
2342-2345
96.
Tandon, N. N.,
Ockenhouse, C. F.,
Greco, N. J.,
and Jamieson, G. A.
(1991)
Blood
78,
2809-2813
97.
Diaz-Ricart, M.,
Tandon, N. N.,
Carretero, M.,
Ordinas, A.,
Bastida, E.,
and Jamieson, G. A.
(1993)
Blood
82,
491-496
98.
Adams, J. C.
(1997)
Int. J. Biochem. Cell Biol.
29,
861-865
99.
Stomski, F. C.,
Gani, J. S.,
Bates, R. C.,
and Burns, G. F.
(1992)
Exp. Cell Res.
198,
85-92
100.
Dorahy, D. J.,
Thorne, R. F.,
Fecondo, J. V.,
and Burns, G. F.
(1997)
J. Biol. Chem.
272,
1323-1330
101.
Ren, Y.,
Silverstein, R. L.,
Allen, J.,
and Savill, J.
(1995)
J. Exp. Med.
181,
1857-1862
102.
Franc, N. C.,
Heitzler, P.,
Ezekowitz, R. A.,
and White, K.
(1999)
Science
284,
1991-1994
103.
Savill, J.,
Dransfield, I.,
Hogg, N.,
and Haslett, C.
(1990)
Nature
343,
170-173
104.
Stern, M.,
Savill, J.,
and Haslett, C.
(1996)
Am. J. Pathol.
149,
911-921
105.
Fadok, V. A,
Warner, M. L,
Bratton, D. L.,
and Henson, P. M.
(1998)
J. Immunol.
161,
6250-6257
106.
Erwig, L. P.,
Gordon, S.,
Walsh, G. M.,
and Rees, A. J.
(1999)
Blood
93,
1406-1412
107.
Coopman, P. J.,
Thomas, D. M.,
Gehlsen, K. R.,
and Mueller, S. C.
(1996)
Mol. Biol. Cell
7,
1789-1804
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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