From the Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, September 21, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Recent literature suggests that tetraspanin
proteins (transmembrane 4 superfamily; TM4SF proteins) may associate
with each other and with many other transmembrane proteins to form
large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with
Transmembrane 4 superfamily
(TM4SF)1 proteins (also
called tetraspanins) comprise a large group of ubiquitously expressed
proteins that function in diverse contexts such as T- and B-cell
activation, platelet aggregation, and cell fusion, motility, and
proliferation (1-3). Although TM4SF molecules often associate with
integrins, TM4SF proteins themselves have little influence on cell
adhesion (4), and these proteins do not localize into focal adhesion complexes (5). The capability of TM4SF molecules to associate with each
other and with a multitude of other molecules suggests that TM4SF
proteins may serve as adaptors involved in the assembly of protein
complexes in the membrane (2, 6). A particularly striking example of
TM4SF protein complexes is the highly organized arrangement of
uroplakins 1A and 1B with other proteins in the urothelial membrane
(7).
TM4SF protein associations can be divided into three general
categories. Level 1 consists of very robust associations, stable in
Triton X-100 detergent, and likely to be direct. Perhaps the best
example is the Level 3 associations are the most numerous and are least likely to be
direct. These complexes are disrupted by Triton X-100, Brij 96, or Brij
97 but may be retained in 1% Brij 99, 1% CHAPS, or other less
hydrophobic detergents. These include TM4SF associations with MHC class
I and class II proteins and other Ig superfamily proteins (21-23),
many additional integrins besides Because level 3 (and to a lesser extent level 2) complexes may contain
large numbers of protein components and are resistant to mild
detergents, it appeared possible that these complexes could be part of
"raft" type plasma membrane microdomains (33-35). In this regard,
association of integrin Rafts differ from other parts of the plasma membrane by their high
content of sphingolipids, cholesterol, and phospholipids with long,
saturated fatty acyl side chains. This unique composition causes lipids
in these microdomains to form a liquid-ordered phase rather than the
conventional liquid-crystalline phase in the rest of the membrane (34).
Thus, many raft-localized molecules are resistant to extraction with
cold nonionic detergents. These insoluble membrane microdomains are
also called detergent-resistant membranes or detergent-insoluble
glycolipid-enriched membranes. The ratio of lipid to protein in these
domains is higher than in surrounding parts of the membrane, enabling
their segregation into the low density (light membrane) fractions of
isopycnic sucrose gradients.
The tendency of TM4SF proteins to associate with each other and with so
many other proteins has created enormous problems in evaluating the
specific biochemical properties of these proteins. The possible
location of these complexes in raft-type microdomains, coupled with the
appearance of level 2 and level 3 complexes only under mild detergent
conditions, suggests that possibly these complexes may exist only in
the context of large and poorly solubilized membrane vesicles. In this
regard, the integrin Here we examine TM4SF complexes in terms of their densities, relative
sizes, and possible relationship to lipid rafts. As prototype level 2 complexes, we have analyzed CD9- Cell Lines and Antibodies Immunoprecipitation and Blotting--
Cells were labeled with
sulfo-N-hydroxysuccinimide-LC-Biotin (Pierce) at 0.1 mg/ml for 30 min at 4 °C, washed with cold PBS containing 200 mM glycine, and lysed with buffer containing 1% detergent
(Triton X-100, CHAPS, Brij 99, or Brij 96), 25 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 20 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride, 2 mM NaF, 10 mM sodium pyrophosphate, and 10 mM Na3VO4. After 1 h at
4 °C, insoluble material was removed by centrifugation at
16,000 × g (10 min, 4 °C), and the supernatant was
cleared with protein G-Sepharose (Amersham Pharmacia Biotech). For
immunoprecipitation, specific antibodies were added for 2 h, and
then protein G-Sepharose was added for another 2 h. Immune complexes were collected by centrifugation, washed four times in lysis
buffer, and then analyzed by SDS-PAGE under nonreducing conditions. For
immunoblotting, proteins resolved by SDS-PAGE were transferred to a
nitrocellulose membrane and then incubated with primary antibodies and
horseradish peroxidase-conjugated secondary antibody (Sigma) as
described (9) or with ExtrAvidin coupled to horseradish peroxidase
(Sigma) to detect biotinylated proteins. Blots were visualized by
chemiluminescence, using the ECL system from Amersham Pharmacia Biotech.
PtdIns 4-K Assay--
PtdIns 4-K assays were performed as
described (55). Briefly, samples from immunoprecipitates or sucrose
gradients were analyzed in a 100-µl final volume containing 0.3%
Triton X-100, 50 µM ATP, 10 mM
MgCl2, 20 mM HEPES (pH 7.5), 0.2 mg/ml
sonicated phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL),
and 5-20 µCi of [ Sucrose Gradients--
Cells were lysed as described for
immunoprecipitations, except that 25 mM MES (pH 6.5)
replaced HEPES. Lysates were then sheared by successive passage through
hypodermic needles (5 × 16G11/2, 10 × 26G1/2). 1 ml of
lysate (derived from 2 × 107 cells) was then mixed
with an equal volume of 90% sucrose. This 45% layer was overlaid with
2 ml of 35% sucrose and 1 ml of 5% sucrose (prepared in MES buffer
without detergent). Samples were centrifuged at 200,000 × g for 16-18 h at 4 °C in a Beckman SW55 rotor, and
fractions of 400 µl each were collected from the top of the gradient.
Also, the pellet was suspended in 400 µl of 25 mM MES,
150 mM NaCl, 5 mM MgCl2, detergent,
and inhibitors. As an alternative, the detergent-free method of Song
et al. (56) was utilized. Briefly, cells were suspended in
buffer (pH 11) containing Na2CO3 (see figure
legends for concentration) plus inhibitors (aprotinin, leupeptin,
phenylmethylsulfonyl fluoride, NaF, sodium pyrophosphate, and
Na3VO4) and lysed by applying 20 strokes in a
loose fitting Dounce homogenizer. Lysates were then sonicated (3 × 20 s), and particulate material was removed by centrifugation
at 16,000 × g for 10 min. Gradients were then carried out as above, except that sucrose layers contained
Na2CO3 and lacked MgCl2. All lysis
and gradient procedures were carried out at 4 °C.
Cholesterol Depletion--
To specifically remove cholesterol,
intact cells were washed three times in PBS to remove serum and then
were incubated in Dulbecco's modified Eagle's medium containing 10 or
20 mM methyl- Gel Chromatography--
Sepharose CL6B columns (Amersham
Pharmacia Biotech; 1 × 18 cm) were equilibrated with lysis buffer
at 4 °C (Brij 99-containing buffers) or at room temperature (Brij 96 lysis), and 0.7 ml of cell lysate (from ~5 × 107
cells) was applied to the top of the column. The column was eluted with
the respective buffer used for cell lysis, and 0.3-0.6-ml fractions
were collected. The column was calibrated with blue dextran and phenol
red to define the void volume and small molecule elution volume.
Immunofluorescence Microscopy--
A431 cells were cultured
overnight on glass coverslips, washed with PBS containing 2 mM MgCl2, and then blocked with 2% bovine serum albumin in PBS/MgCl2 for 10 min at room temperature.
Cells were then incubated with primary antibody (at 10 µg/ml, in
Dulbecco's modified Eagle's medium containing 50 mM
HEPES, 2 mM MgCl2, and 2% bovine serum
albumin) for 30 min at 4 °C. After three washes with
PBS/MgCl2, cells were fixed in 3% paraformaldehyde (30 min at room temperature), washed three times again, and then incubated with
secondary antibody (5 µg/ml AlexaFluor488 goat anti-mouse IgG;
Molecular Probes, Inc., Eugene, OR) for 30 min at 4 °C. After three
washes, GM1 ganglioside was detected by adding biotinylated B-subunit
of cholera toxin (CT-B, 10 µg/ml; Sigma) and then NeutrAvidin-Texas Red (5 µg/ml; Molecular Probes). After another washing step,
coverslips were mounted onto glass slides using the ProLong Antifade
Kit from Molecular Probes. In a separate experiment, the sequence of
incubations was changed such that cells were first incubated with
biotinylated CT-B and fixed, and then NeutrAvidin-Texas Red and primary
and secondary antibodies were added. Slides were analyzed using a Zeiss
Axioskop, with an HBO 100-watt mercury lamp. Photographs were obtained
using a Dage-MTI CCD camera, and processed with Scion Image software
and Adobe Photoshop. The AlexaFluor488 dye emitted essentially no
fluorescence when samples were irradiated with the Texas Red excitation
wavelength and vice versa. Incubation with secondary
antibody or NeutrAvidin-Texas Red alone led to negligible cell staining.
CD9-
Several additional proteins, not identified here, were also present in
both the CD9 and CD9 and
In contrast, in 1% CHAPS, substantial amounts of
Because detergents such as CHAPS or Brij 99 could artificially induce
cell surface protein complex formation in the light membrane fractions,
we utilized an alternative detergent-free isolation method developed by
Song et al. (56). Results obtained using this high pH
"carbonate" method (Fig. 2D) were similar to those
obtained using 1% CHAPS (Fig. 2B). Substantial amounts of CD9 and
To further address the raftlike nature of CD9 and CD9-
The disappearance of CD9 and
To further test the effect of cholesterol depletion on TM4SF protein
and integrin complexes, cell surface biotinylated proteins from A431
cells were immunoprecipitated, and then half of each complex was
incubated with 20 mM M CD9-
To demonstrate further that CD9-
Although caveolin-1 colocalized with
Besides cholesterol, the actin cytoskeleton may also markedly influence
the localization of proteins into rafts (62) and the distribution of
TM4SF protein complexes (5). However, when cells were treated with
latrunculin B (10 µM) before lysis to disrupt actin
filaments, we observed no change in the association of CD9 with
CD81-
Comparison of CD81 immunoprecipitates (Fig. 8, middle and
bottom panels) with total biotin-labeled cell surface
protein (top panel) revealed that only a minority
of total labeled protein was in the CD81 complexes. Also, with the aid
of additional sucrose layers to increase resolution, we observed that
CD81- How Big Are TM4SF- Analysis Of CD63-PtdIns 4-K as a Prototype Level 3 Complex--
Because TM4SF "level 3" interactions may include so
many different molecules, we hypothesized that many of these
interactions were especially likely to result from indirect
associations within large lipid microdomains. Sensitivity to Brij 96 distinguishes PtdIns 4-K-CD63 protein complexes (level 3) from
Upon sucrose gradient analysis of 1% Brij 99 lysates, a substantial
fraction of CD63 appeared in light membrane fractions (Fig.
7A, left panel). Substantial PtdIns
4-K activity from 1% Brij 99 or 1% CHAPS lysates was also present in
the light membrane fractions, whereas 1% Triton lysates yielded
essentially no PtdIns 4-K activity in the light membrane fractions
(Fig. 10). Immunoprecipitation analysis
of 1% Brij 99 sucrose gradient fractions revealed that PtdIns 4-K
activity was associated with CD63 in both light membrane and dense
fractions (Fig. 11, A and
B). Furthermore, under conditions (1% Brij 99 plus 0.2%
Triton X-100) in which CD63 was almost entirely present in dense
fractions (Fig. 7A, right panel),
PtdIns 4-K association was again well maintained (Fig. 11C).
Little PtdIns 4-K association with negative control proteins
(
TM4SF-PtdIns 4-K association did not depend on cholesterol. Treatment
of immunoprecipitated CD63, CD9, and
Finally, CD63-PtdIns 4-K complexes were fractionated using a CL6B gel
filtration column to allow a rough estimate of complex size. From a
Brij 99 lysate of A431 cells, a portion of the CD63-associated PtdIns
4-K activity was partly excluded from the column (Fig. 12A, fractions
1 and 2), whereas another portion was included
well within the column (Fig. 12A, fractions
5-10). If 0.2% Triton X-100 was included with the Brij 99, the yield of CD63-associated PtdIns 4-K was diminished, but the
majority of the remaining activity migrated well within the included
volume of the column (Fig. 12B). In both detergent
conditions, a substantial fraction of the total PtdIns 4-K activity
migrated in fractions 10-18, indicating that it was smaller in size
and mostly dissociated from CD63. From these results, we conclude that
CD63-PtdIns 4-K complexes can exist at a size considerably less than
4 × 106 Da.
Prototype TM4SF Protein Complexes Occur as Discrete
Units--
TM4SF proteins have been reported to associate with each
other, with integrins, and with many other types of transmembrane proteins and other proteins, thus potentially forming an extensive network (1, 3, 6, 63). Many of these associations are best seen when
weak (i.e. less hydrophobic) detergents are utilized. Thus,
a major concern regarding many TM4SF protein complexes has been that
they may represent incompletely solubilized vesicular material. These
complexes may sometimes be very large (>20 million Da (49)) and not
very dense (37, 39), consistent with incomplete solubilization and a
high lipid/protein ratio. Indeed, our initial results showed that the
detergent conditions most permissive for TM4SF associations with other
proteins (e.g. 1% Brij 99, 1% CHAPS) were also most likely
to yield a large fraction of TM4SF proteins and associated proteins
within the incompletely solubilized light membrane fractions of a
sucrose gradient.
However, our studies of a few prototype TM4SF protein complexes now
demonstrate that (a) these complexes are well maintained within the detergent solubilized dense fractions of sucrose gradients, (b) they are not dependent on cholesterol for maintenance of
association, and (c) they can occur well within the included
volume of a Sepharose CL6B gel filtration column. Also we have
confirmed that these complexes are indeed highly specific in terms of
the components present in the complex. Together, these results
emphasize that these complexes are not artifacts of incomplete
solubilization but instead represent discrete units that are of
moderate size and capable of being fully solubilized.
Association of
Neither CD9-
Incompletely solubilized low density fractions from sucrose gradients
typically contain large vesicles of 0.05-1 µm in diameter (65, 66).
However, complexes of CD9- Prototype TM4SF Protein Complexes in Lipid Raftlike
Domains--
Although not dependent on a lipid raftlike environment
for association, nonetheless we establish here that prototype TM4SF protein complexes may localize into raft-type microdomains. First, under multiple detergent lysis conditions,
CD9-
Previously, CD9 was suggested to be present in T cell rafts (in Triton
X-100 conditions), where it could perhaps facilitate T cell
costimulation (39). Here we have confirmed the raftlike association of
CD9 (and CD9 complexes), although in our hands this was not very
obvious using Triton X-100 conditions. Instead, we needed to utilize
Brij 99, CHAPS, or detergent-free conditions to see appreciable CD9 in
the light membrane fractions of sucrose gradients. Also, our
CD63-PtdIns 4-K raft-type localization results are consistent with
previous reports that the PtdIns 4-K enzyme, as well as PtdIns 4-K
lipid substrate (PtdIns) and product (PtdIns 4-P) are in rafts (42,
67). The Different Types of Rafts--
An abundance of evidence now
suggests the existence of distinct types of lipid microdomains, each
with specific components. For example, microdomains containing caveolin
are distinct from other types of microdomains (42, 66, 72, 73). Also,
microdomains may differ in terms of types of gangliosides that are
present (72), the presence of particular GPI-linked proteins (65), and
the presence or absence of other specific cell surface proteins (74).
Integrin association with caveolin (60, 61) and the GPI-linked protein
uPAR (60) might suggest that these known raft-associated molecules
could facilitate the recruitment of integrin complexes into raftlike
domains. However, the TM4SF protein complexes studied here are present
in a distinct type of domain, not containing caveolin, uPAR, or other
GPI-linked proteins. Using conditions in which we see
TM4SF-
Another means of subdividing rafts is by detergent solubility (65, 74).
Rafts were originally defined as being Triton-insoluble (33), but
subsequent data have emphasized that distinct types of rafts may differ
markedly in terms of detergent solubility. In our studies, detergents
such as Brij 96 and Brij 99 were better than Triton X-100 in
maintaining TM4SF complexes in raftlike domains. Notably, microdomains
containing
According to one model, different types of rafts may be distinct and
nonadjacent (74). Alternatively, there may be a continuum of highly
ordered detergent insoluble rafts, in proximity to semiordered domains
that are more soluble in detergent (65). Such a continuum of
detergent-insoluble rafts adjacent to less ordered rafts could explain
our observation that CD9 and Functional Relevance--
Rafts have broad functional relevance
with respect to signaling and protein sorting (33, 34, 76). However, in
the absence of transmembrane proteins, it has been difficult to
visualize how rafts can act as concentrated signaling loci. Although
TM4SF proteins are not very tightly associated with the cytoskeleton (consistent with their ease of extraction by detergents), the actin
cytoskeleton does appear to influence TM4SF protein distribution (5).
We propose that TM4SF-containing microdomains, by being in proximity to
highly ordered raftlike domains, could help regulate the distribution
and function of rafts. For example, TM4SF protein localization into
cell filopodia or microvilli (77, 78) could potentially influence the
distribution of rafts to these same locations. Conversely, TM4SF
protein complexes may also help to recruit signaling enzymes into the
proximity of rafts. For example, TM4SF proteins complexed with PtdIns
4-K may bring this enzyme into proximity with rafts that are known to
be enriched in PtdIns lipid substrate (67). Likewise, by
associating with rafts, TM4SF complexes may come into proximity with
raft signaling components, such as Src family kinases and G proteins
(44, 73).
Studies elsewhere point to a potential functional role for TM4SF
protein complexes in rafts. For example, costimulation of T lymphocytes
clearly involves reorganization of rafts (79), and CD81 and CD9 both
play a role in T cell costimulation (6, 80). Also, a possible role for
TM4SF protein complexes in epidermal growth factor receptor-containing
rafts may be inferred because (a) the epidermal growth
factor receptor associates with both TM4SF proteins (81) and PtdIns 4-K
(82), (b) TM4SF-PtdIns 4-K complexes localize into rafts (as
shown here), and (c) activation of the epidermal growth
factor receptor may occur within raft-type microdomains that are
distinct from caveolin-rich domains (83). In other studies, rafts (84,
85), TM4SF proteins (16, 86, 87), and PtdIns 4-K (88, 89) have all been
implicated in vesicular trafficking events, thus supporting speculation
that TM4SF complexes in rafts may be playing a key role.
In conclusion, we have utilized a few prototype TM4SF complexes to
establish that TM4SF complexes may exist as well solubilized discrete
units, apart from lipid raftlike microdomains. Nonetheless, these
complexes also may associate with microdomains that resemble lipid
rafts by several criteria. Thus, we now have a mechanistic basis to
begin explaining how TM4SF proteins and lipid raft-type microdomains in
general may be involved in many overlapping functions.
3
1 (an integrin) and
complexes of CD63 (a TM4SF protein) with phosphatidylinositol
4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like
microdomains, as seen by three different criteria. First, these
complexes localize to low density light membrane fractions in sucrose
gradients. Second, CD9 and
3 integrin colocalized with
ganglioside GM1 as seen by double staining of fixed cells. Third,
CD9-
3
1 and CD81-
3
1 complexes were shifted to a higher
density upon cholesterol depletion from intact cells or cell lysate.
However, CD9-
3
1, CD81-
3
1, and CD63-PtdIns 4-K complex
formation itself was not dependent on localization into raftlike lipid
microdomains. These complexes did not require cholesterol for
stabilization, were maintained within well solubilized dense fractions
from sucrose gradients, were stable at 37 °C, and were small enough
to be included within CL6B gel filtration columns. In summary,
prototype TM4SF protein complexes (CD9-
3
1, CD81-
3
1, and
CD63-PtdIns 4-K) can be solubilized as discrete units, independent of
lipid microdomains, although they do associate with microdomains
resembling lipid rafts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1-CD151 complex (8, 9).
Other possible direct associations include the
6
1-CD151, and
4
1-CD81 complexes (10). Level 2 associations are more numerous but nonetheless highly specific. These
are disrupted by 1% Triton X-100 but are retained in 1% Brij 96 or
Brij 97 detergent and other less hydrophobic detergents. These include
complexes containing any of several different TM4SF proteins (such as
CD9, CD63, CD81, CD82, CD151, or NAG-2), linked to each other, to a
subset of integrins (
3
1,
6
1), or to other transmembrane proteins
(11-19). In several experiments, antibodies to both TM4SF proteins and
associated integrins could similarly inhibit cell motility or neurite
outgrowth (8, 15, 16, 20). These results support the functional
relevance of some level 1 and level 2 TM4SF complexes.
3
1 and
6
1 (16, 24-26), proteoglycans (27), and
various signaling molecules (28-32).
V
3 with CD47, a
member of the immunoglobulin superfamily with multiple transmembrane domains, is cholesterol-dependent and occurs in a lipid
raft environment (36). Likewise,
1 integrins (37),
integrin
L
2 (38), and TM4SF protein CD9
(39) may localize in lipid raft domains. Furthermore, CD4 (40), CD36
(41), type II phosphatidylinositol 4 kinase (PtdIns 4-K) (42), various
GPI-linked proteins (43, 44), and conventional protein kinase C
isoforms (45) each have also been suggested to localize into raftlike
domains. These same proteins including CD4 (46), CD36 (37), PtdIns 4-K
(8, 30, 31), various GPI-linked proteins (47), and conventional protein
kinase C (48) proteins associate with TM4SF proteins and/or integrins. Thus, we hypothesize that TM4SF-integrin plasma membrane complexes may,
at least to some extent, resemble rafts.
L
2-CD63 complex was
excluded from a Sepharose CL4B column, suggesting a size equal to or
greater than 20 million daltons (49).
3
1 (12, 14) and in some experiments CD81-
3
1 (12,
15, 20) associations. As a prototype level 3 complex, we analyzed
CD63-PtdIns 4-K association (30, 31). In each case, we asked whether
these complexes only exist in the context of large raftlike vesicles or
whether they may exist in a truly soluble form of a reasonable size.
These studies have been carried out using A431 and HT1080 cell lines because they express significant levels of
3
1, CD9, and/or CD81. Also, A431 cells
were selected for study because they were used previously to establish
that PtdIns 4-K may localize into lipid rafts (42).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
Fibrosarcoma cell line HT1080
and epidermoid carcinoma line A431 were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum and
antibiotics. Monoclonal antibodies used were anti-integrin
2, A2-IIE10 (50); anti-integrin
3, A3-IVA5 (51); anti-integrin
1, TS2/16 (52); anti-CD9,
C9-BB (12) and DU-ALL-1 (Sigma); anti-CD63, 6H1 (11); anti-CD81, M38
(53); anti-CD151, 5C11 (8); anti-CD71, OKT9 and HB21 (American Type
Culture Collection) and DF1513 (Sigma); anti-E-cadherin, C20820
(Transduction Laboratories, Lexington, KY); and anti-caveolin-1, C13630
(Transduction Laboratories). Also utilized was a rabbit polyclonal
antibody to the cytoplasmic domain of integrin
3A (54).
-32P]ATP. For time and
temperature, see legends. Assays were stopped by adding 5 N
HCl (25 µl), and the lipids were extracted with 160 µl 1:1
chloroform/methanol (v/v). The organic phase was collected and
separated by thin layer chromatography. TLC plates were scanned with a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA), and the pixel
intensities of spots corresponding to radiolabeled PtdIns 4-P were
determined with ImageQuant software (Molecular Dynamics).
-cyclodextrin (M
CD) for 30-60 min at
37 °C. The sample was then successively centrifuged at 200, 2000, and 16,000 × g to remove cells and cellular debris
from the sample. For treatment of immunoprecipitated samples, immune
complexes on Sepharose beads were incubated in lysis buffer without
detergent but containing 10 mM M
CD for up to 60 min at 4 or 37 °C. Levels of cholesterol in lysates and cell-free
supernatants of M
CD-treated cells were determined using the
"Cholesterol 20" kit (Sigma). This kit utilizes cholesterol oxidase
to produce hydrogen peroxide, which is then detected in a coupled
colorimetric peroxidase assay. Ganglioside GM1 was measured in a dot
blot assay, utilizing cholera toxin B subunit, anti-cholera toxin
polyclonal antibody, and HRP-conjugated anti-rabbit antibody (all
reagents from Sigma).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1 Complexes under Different
Detergent Conditions--
A key feature of lipid rafts is their
relative resistance to detergents (33, 34). To examine the detergent
resistance of TM4SF-integrin protein complexes, A431 human carcinoma
cells were surface labeled with biotin and lysed using four different detergent conditions, and then immunoprecipitations were carried out.
In 1% Brij 99 detergent, immunoprecipitation of
3
1 integrin yielded a protein comigrating
with CD9, and immunoprecipitation of CD9 yielded
3
1 integrin. Both proteins appeared at
nearly the same level regardless of which antibody was utilized,
consistent with a high stoichiometry of complex formation. In 1%
CHAPS, similar results were obtained, except that less
3
1 was associated with CD9 and less CD9
was associated with the immunoprecipitated
3
1. In contrast, under conditions of 1%
Triton X-100 or RIPA (1% Triton X-100, 0.5% deoxycholate, 0.1% SDS),
essentially no CD9-
3
1 complex was
observed. The identity of
3
1 in CD9
immunoprecipitations and CD9 in
3 immunoprecipitations
has been demonstrated numerous times by reimmunoprecipitation and/or by
immunoblotting (Refs. 12 and 14 and data not shown).
3
1 immunoprecipitated
complexes. The levels of these other proteins progressively diminished
as conditions were shifted from 1% Brij 99 to 1% CHAPS to 1% Triton X-100 to RIPA. The prominent ~85-kDa protein and more weakly labeled 70-kDa protein seen in
3 immunoprecipitations
(especially in Triton and RIPA conditions) probably arise from
3 proteolysis as characterized elsewhere (57). In
control experiments, the
2
1 integrin was
immunoprecipitated in the absence of any apparent CD9 association, and
E-cadherin immunoprecipitates showed no evidence for either CD9 or
integrin association, regardless of detergent conditions.
3 Integrin Appear in Raftlike Membrane
Microdomains--
Because of the types of proteins sometimes found to
associate with TM4SF proteins and integrins (see Introduction), and
from results such as those shown in Fig.
1, we hypothesized that
3
1-CD9 complexes might occur in raftlike
microdomains that would be maintained in Brij or CHAPS conditions but
disrupted in 1% Triton. Indeed, sucrose density gradient analysis of a
1% Triton lysate revealed that only a trace of CD9 or
3
integrin (<1%) was present in the low density, lipid-enriched, light
membrane fractions (Fig. 2A, lanes 3-5). As expected, the majority of total
cell surface biotin-labeled proteins (top panel)
and E-cadherin (bottom panel) were in the dense
fractions (lanes 7-12), while caveolin-1 (well
known to appear in Triton-insoluble vesicular microdomains (41)) served as a positive control for the light membrane fraction.
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Fig. 1.
Coimmunoprecipitation of
3 integrin, CD9, and associated
proteins under different detergent conditions. A431 cells were
trypsinized, surface-biotinylated, and then lysed using the indicated
detergents at 1%. Immunoprecipitations (IP) were
performed using mAb A2-IIE10 (
2), A3-IVA5
(
3), DU-ALL-1 (CD9), and C20820 (E-cadherin), and
proteins were separated by SDS-PAGE, using a linear 6-20% gel
gradient, under nonreducing conditions. After blotting onto
nitrocellulose, precipitated proteins were detected by incubation with
ExtrAvidin-HRP. The major protein observed upon E-cadherin
immunoprecipitation (~50 kDa) may correspond to a previously
described major E-cadherin degradation product (90)
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Fig. 2.
Distribution of
3
1,
CD9, and control proteins in isopycnic sucrose gradients.
Surface-biotinylated A431 cells were lysed (2 × 107
cells in 1 ml) using either 1% Triton X-100 (TX-100)
(A), 1% CHAPS (B), 1% Brij 99 (C),
or 150 mM Na2CO3 (pH 11) with no
detergent (D). Lysates were then centrifuged in a
discontinuous sucrose density gradient, 12 fractions of 400 µl each
were collected from the top of the gradient, and the pellet was
resuspended in an additional 400 µl of lysis buffer. 10 µl of each
fraction was applied to nonreducing SDS-PAGE, and immunoblotting was
carried out using antibodies against caveolin-1 (C13630), CD9 (C9-BB),
3 integrin (rabbit polyclonal anti-C terminus), and
E-cadherin (C20820). Total surface-biotinylated material was detected
by incubation with ExtrAvidin-HRP. The identity of the major
biotinylated protein appearing in the light membrane fractions
(top panels) is unknown. Light membrane fractions
3-6 (indicated by brackets) occur near the 5/35% sucrose
interface. Sizes of molecular weight markers are indicated in the
top panel. For the lower
four panels, approximate sizes of caveolin-1 (22 kDa), CD9 (24 kDa), integrin
3 (150 kDa), and E-cadherin
(120 kDa) are indicated in parentheses.
3
integrin and CD9 appeared in the light membrane fractions, as did the positive control protein, caveolin-1 (Fig. 2B). Again, the
majority of E-cadherin and total biotin-labeled proteins were in the
dense fractions. Similarly in 1% Brij 99, large amounts of CD9,
3 integrin, and caveolin were present in the light
membrane fractions (Fig. 2C), while larger proportions of
total biotin-labeled proteins and E-cadherin also appeared in the light
membrane fractions (Fig. 2C). Despite the increased
proportion of E-cadherin in light membrane fractions in Brij 99 conditions, it did not show any coimmunoprecipitation with
3 or CD9 (Fig. 1). Also,
2 integrin was
present in light membrane fractions (not shown) although not associated
with CD9 or
3 integrin. Thus, as many others have noted,
components can appear together in light membrane fractions without
necessarily being associated.
3 integrin appeared in the light membrane
fractions, while most of the E-cadherin and total biotin-labeled
protein were present in the dense fractions.
3
integrin complexes, GM1 ganglioside colocalization studies were carried out, using cholera toxin as a probe for GM1. A431 cells were first incubated with biotinylated CT-B, and then after cells were
fixed, CT-B was visualized (Fig.
3A, right
panels). In the same experiment, fixed cells were also
double-stained for CD9,
3 integrin, or CD71 (transferrin
receptor) (Fig. 3A, left panels). As
indicated, CD9 and
3 integrin each showed substantial
colocalization with CT-B-stained GM1 ganglioside, whereas CD71 staining
was largely nonoverlapping with the CT-B staining. A reciprocal double
staining experiment was also carried out (Fig. 3B). Spread
A431 cells were first incubated with anti-CD9, anti-
3,
or anti-CD71 mAb, and then after fixing, those proteins were visualized
(Fig. 3B, left panels). Double
staining was then carried out using biotinylated CT-B (Fig.
3B, right panels). Again, staining of
CD9 or
3 integrin showed substantial overlap with CT-B
staining, whereas staining for CD71, a nonraft protein (44), was quite
distinct. Colocalization of CD9 and
3
1
was readily observed (Fig. 3, A and B) when cells were fixed immediately after staining with primary antibody; it was not
necessary to induce further clustering of the antigens by adding second
antibody or multivalent anti-CT-B reagents.
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Fig. 3.
Codistribution of GM1 with TM4SF complexes on
A431 cells. A, A431 cells were labeled with antibodies
against 3 integrin (A3-IVA5), CD9 (DU-ALL-1), or CD71
(DF1513). After fixing in 3% paraformaldehyde, cells were incubated
with Alexa488-coupled rabbit-anti mouse-IgG secondary antibody and then
double-stained using biotinylated cholera toxin-B (CT-B) and
NeutrAvidin-Texas Red. B, A431 cells were labeled with
biotinylated CT-B, fixed in paraformaldehyde, and then incubated with
NeutrAvidin-Texas Red and double-stained using anti-
3,
CD9, or CD71 antibody, followed by Alexa488-coupled secondary antibody.
In both experiments, Texas Red staining of CT-B is shown in the
right panels, and Alexa488 staining of antibodies
is shown in the left panels. The AlexaFluor488
dye emitted essentially no fluorescence when samples were irradiated
with the Texas Red excitation wavelength and vice versa.
Scale bars, 10 µm.
3
1 Complexes Are Perturbed upon
Cholesterol Depletion but Do Not Require Cholesterol for
Association--
Partial depletion of cholesterol typically leads to a
loss of protein localization into rafts (44).
-Cyclodextrins are a
class of heptasaccharides commonly used to selectively remove cholesterol from cellular membranes (58). By treating intact A431 cells
with M
CD, we typically removed 40-60% of total cholesterol, as
measured following cell lysis. After M
CD treatment of intact cells,
we then analyzed CD9 and
3 integrin appearance in light membrane fractions, following isopycnic sucrose centrifugation using
the detergent-free carbonate lysis method. As indicated in Fig.
4, the amounts of CD9 and
3 in light membrane fractions were greatly diminished
compared with untreated control samples run in parallel. As expected,
levels of caveolin in light membrane fractions were also diminished
upon cholesterol depletion. Results in Fig. 4 are again consistent with
CD9-
3 integrin complexes being present in raftlike
microdomains.
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Fig. 4.
Cholesterol depletion results in
diminished 3 and CD9 in light
membrane fractions of sucrose density gradients. A431 cells were
serum-starved for 24 h and surface-biotinylated, and the intact
cells were incubated in the presence of 10 mM M
CD for 30 min at 37 °C. Cells were then scraped into 150 mM
Na2CO3 and lysed by Dounce homogenization and
sonication (without detergent), and sucrose density gradients were
prepared. From each sucrose gradient fraction, 10 µl was resolved by
SDS-PAGE, and the indicated antigens were detected by Western blotting.
Light membrane fractions 4-6 (indicated by brackets) occur
near the 5/35% sucrose interface.
3 integrin from light
membrane fractions (lanes 3-6) was not
accompanied by a large increase in these proteins in the dense
fractions (Fig. 4, lanes 8-12). Thus, we looked
for shed proteins in the M
CD-treated cell supernatant. Immunoprecipitations were carried out from cell-free supernatants of
M
CD-treated A431 cells in the presence of 1% Brij 99. As indicated (Fig. 5A), shed complexes
immunoprecipitated by antibodies to
3
1,
CD9, or CD151 (a TM4SF protein tightly associated with
3
1 (9)) resembled complexes directly
immunoprecipitated from whole cell lysates (e.g. Figs. 1 and
6) and thus appeared to remain intact.
Control protein CD71 was present in the cell-free supernatant but was
not part of the TM4SF-
3
1 complex (Fig.
5A). Control proteins caveolin and E-cadherin were not
present in the shed fraction (not shown). In the absence of M
CD
treatment, little protein was found in the shed fraction (Fig.
5B), thus establishing that shedding was indeed induced by
M
CD, as seen previously for other protein complexes (59). Also, as
seen previously (59), the ganglioside GM1 was present in shed membrane
vesicles. As determined by an immunochemical assay involving cholera
toxin B subunit, the amount of GM1 shed into the cell-free supernatant of M
CD-treated A431 cells was about 8 times higher than that of
untreated samples (data not shown). In contrast, cholesterol was not
present in shed membrane vesicles. After M
CD treatment, shed
vesicles were isolated by centrifugation (200,000 × g
for 1 h), and the pellet was washed once with PBS. Cholesterol
measurements showed no difference between material isolated from
cell-free supernatants of M
CD-treated or -untreated cells. In fact,
all cholesterol present in M
CD-treated cell supernatants could be completely removed by dialysis, as expected for
cholesterol-cyclodextrin complexes (data not shown). Sucrose gradient
analysis of shed fractions confirmed that after cholesterol depletion,
CD9 and
3 integrin complexes were no longer present in
light membrane fractions (Fig. 5C, bottom
panels, lanes 3-5), although they
were perhaps slightly less dense than the total biotin-labeled protein (Fig. 5C, top panel). Thus,
cholesterol depletion perturbed CD9-
3
1 complexes insofar as inducing shedding and increasing their density, but the protein complexes themselves remained intact.
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Fig. 5.
Analysis of TM4SF complexes shed upon
M CD treatment. A431 cells were
trypsinized and surface-biotinylated, and then intact cells were
incubated with 20 mM M
CD for 30 min at 37 °C. The
cell suspension was then cleared of cells and other particulate
material by sequential 10-min centrifugations at 200, 2000, and
16,000 × g. A, the remaining cell-free
supernatant was then adjusted to 1% Brij 99, and immunoprecipitations
were performed using anti-CD9 (DU-ALL-1), anti-
3
(A3-IVA5), anti-CD151 (5C11), and anti-CD71 (DF1513) antibodies. The
immune complexes were resolved by nonreducing SDS-PAGE, and
biotinylated proteins were detected with ExtrAvidin-HRP. B,
M
CD was omitted from A431 cell incubation media, and then cell-free
supernatant was obtained, and immunoprecipitations were carried out as
in A. The arrows represent positions of
3,
1, and CD9 proteins, respectively.
C, cell-free supernatant (obtained as in A) was
adjusted to 100 mM Na2CO3,
homogenized, sonicated, and then subjected to sucrose gradient analysis
as in Fig. 2C. Each fraction was tested for its content of
total biotinylated material (ExtrAvidin-HRP), CD9 (C9-BB), and
3 integrin (polyclonal antibody) by Western blotting.
There was no detectable pellet material from this gradient.
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Fig. 6.
Effects of depletion of cholesterol from
3 integrin and CD9 immune
complexes. A431 cells were trypsinized, surface-biotinylated,
and lysed in 1% Brij 99. Immunoprecipitations were then carried out
using mAbs A2-IIE10 (
2), A3-IVA5 (
3),
DU-ALL-1 (CD9), C20820 (E-cadherin), and 6H1 (CD63). One half of each
precipitated immune complex (immobilized on Sepharose beads) was
incubated with 10 mM M
CD in lysis buffer without
detergent for 2 h at 4 °C on a rotary shaker, and the other
half was mock-treated. After four washes with lysis buffer and one wash
with 20 mM HEPES, 10 mM MgCl2, the
M
CD-treated and -untreated samples were resolved by nonreducing
SDS-PAGE, and cell surface proteins were visualized by blotting with
ExtrAvidin-HRP. For CD63, a longer exposure of the blot is shown.
CD (Fig. 6, + lanes).
As indicated, treatment with M
CD clearly did not alter the levels of
3
1 associated with CD9, or CD9 associated
with
3
1. Likewise, levels of all other
associated proteins were not obviously altered, except for an unknown
protein of ~18 kDa that was substantially diminished upon cholesterol
depletion. Antibodies to another TM4SF protein, CD63, also precipitated
complexes (containing
3
1 and CD9) that were not affected by M
CD treatment. E-cadherin (not shown) and
2 integrin and complexes were likewise unaltered.
Treatment of cell lysates with M
CD or 10 µg/ml filipin (another
cholesterol-disrupting agent) prior to immunoprecipitation again failed
to alter the association of CD9 or CD63 with
3 integrins
and other proteins (not shown).
3
1 Complexes Readily Occur
Outside of Raftlike Membrane Vesicles--
We next asked whether, in
the absence of cholesterol depletion,
CD9-
3
1 complexes would preferentially
occur in the light membrane fractions from sucrose gradients. As
indicated in Fig. 7A
(left panel), both
3 integrin and
CD9 from 1% Brij 99 lysates were present in light membrane fractions
(lanes 2-5) as well as in dense fractions
(lanes 9-12). As indicated by
immunoprecipitation analysis of sucrose gradient fractions,
CD9-
3 complexes were present in both the dense fractions
(fractions 9-12) and the light membrane fractions (fractions 9-12)
and appeared similar in each location (Fig. 7B,
lanes a-c).
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Fig. 7.
CD9- 3
1
complexes are retained in the dense fractions of sucrose
gradients. A, A431 cells were lysed in either 1% Brij
99 or 1% Brij 99 plus 0.2% Triton X-100, and then sucrose density
gradients were carried out as described in Fig. 2. (Note: mixtures of
detergents were preincubated overnight at 4 °C to form mixed
micelles.) Next, 10 µl of each fraction was applied to nonreducing
SDS-PAGE, and immunoblotting was carried out using antibodies against
caveolin-1 (C13630),
3 integrin (rabbit polyclonal
anti-C terminus), CD9 (C9-BB), CD63 (6H1), and CD71 (mixture of OKT9
and HB21). Protein sizes are ~22 kDa (caveolin-1), ~150 kDa
(integrin
3), ~24 kDa (CD9), 55 kDa (CD63), and ~195
kDa (CD71/transferrin receptor). B, pooled light membrane
fractions (2-5) or dense fractions (9-12) from
the sucrose gradients were diluted 1:5 in lysis buffer and subjected to
immunoprecipitation using mAbs DU-ALL-1 (CD9), A3-IVA5
(
3), A2-IIE10 (
2), and OKT9 (CD71).
Lanes marked by an asterisk were exposed about 5 times longer than the others to compensate for different signal
intensities.
3
1
complexes may occur outside of raftlike domains, we devised more
stringent cell lysis conditions in which these molecules would be
absent from the light membrane fractions but would remain associated.
As seen in Fig. 7A (right panel), the
inclusion of 0.2% Triton X-100 with 1% Brij 99 caused CD9 and
3, but not caveolin, to essentially disappear from the
incompletely solubilized light membrane fractions of a sucrose gradient
and appear exclusively in the dense fractions. Nevertheless,
immunoprecipitation revealed that the CD9-
3 complex was
fully maintained (Fig. 7B, lanes f and
g) and was indistinguishable from the complexes seen in 1%
Brij 99 (lanes a-c). In control experiments,
anti-
2 integrin did not coimmunoprecipitate CD9, and
anti-CD71 did not coimmunoprecipitate integrins or CD9 (Fig. 7B, lanes d, e,
h, and i).
3
1
and CD9 in the light membrane fractions of some sucrose gradients
(Figs. 2, 4, and 7A) and interactions between caveolin-1 and
1 integrins have been suggested (60, 61), we could not
coprecipitate caveolin-1 with either CD9 or
3 integrin
in Brij 99 conditions. Furthermore, we have previously observed
3
1-CD9 complexes in the K562 cell line
(12), which does not express caveolin-1.
3
1 (not shown). Another key feature of
detergent-resistant raft complexes in vitro is their
sensitivity to increased temperature (33, 34). In this regard, our
TM4SF-
3
1 complexes (such as seen in Figs.
6 and 7B) were completely stable despite incubation of cell
lysates for 30 min at 37 °C prior to immunoprecipitation (not shown).
3
1 Complexes Also Occur
Independent of Raftlike Microdomains--
To expand the generality of
our findings, we next analyzed TM4SF complexes using a different cell
line (HT1080), a different TM4SF protein (CD81), and a detergent (Brij
96) that is a little more stringent than Brij 99 or CHAPS with respect
to TM4SF complexes. After sucrose density gradient fractionation,
anti-CD81 immunoprecipitation revealed the presence of
CD81-
3
1 complexes in both light membrane fractions (Fig. 8, middle
panel, lanes 1-3) and in dense
fractions (lanes 4-10). The identity of
3
1 in CD81 complexes was confirmed many
times by reimmunoprecipitation or by immunoblotting (Refs. 12 and 20
and not shown). Upon treatment of the cell lysate with M
CD to
deplete cholesterol prior to density gradient fractionation, CD81-
3
1 complexes in the light membrane
fractions disappeared (lanes 1-3) but were
maintained in the dense fractions (Fig. 8, lower
panel). These results establish again, for another prototype level 2 complex (CD81-
3
1), that such
complexes may localize to raftlike microdomains but do not depend on
this localization for integrity of the complex.
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Fig. 8.
Sucrose gradient analysis of CD81
complexes. Surface-biotinylated HT1080 cells were lysed in 1%
Brij 96, and then without shearing, 800 µl of the lysate (containing
~1 × 107 cells) was centrifuged in a discontinuous
sucrose density gradient containing layers of 50% (0.5 ml), 45% (2 ml), 40% (1 ml), 20% (1 ml), and 5% (0.5 ml). 14 fractions of 360 µl each were collected from the top of the gradient, and the pellet
was included in the 14th fraction. Top panel, 20 µl of each fraction was resolved by SDS-PAGE, and then total
surface-biotinylated proteins were revealed by blotting with
ExtrAvidin-HRP. Middle and bottom
panels, from 300 µl of each fraction, CD81 was
immunoprecipitated, samples were resolved by SDS-PAGE under nonreducing
conditions, and CD81-associated proteins (plus CD81 itself) were
detected by blotting with ExtrAvidin-HRP. For the experiment in the
bottom panel, immunoprecipitation was carried out
as above, except the lysate (800 µl) was treated with 10 mM M CD to deplete cholesterol prior to sucrose gradient
fractionation and immunoprecipitation.
3
1 complexes (bottom
panel) were slightly less dense than the bulk of the total labeled proteins (top panel). Distinct complexes
between CD81 and unknown proteins of ~60 and 200 kDa were also
resolved. The CD81-200-kDa complex (peak, lanes
10-14) was more dense than
CD81-
3
1 (peak, lanes
7-10), whereas the CD81-60-kDa complex (peak,
lanes 5 and 6) was less dense.
3
1
Complexes?--
It was shown elsewhere that TM4SF-integrin complexes
could be rather large (>20 × 106 daltons (49)), thus
raising concerns regarding specificity and complexity. To address the
issue of size, gel filtration was carried out using Sepharose CL6B
columns and 1% Brij 96 (level 2) cell lysis conditions. As indicated,
surface-biotinylated CD9-
3
1 complexes
from A431 cells were readily retained within the included volume of the
column, where they comigrated with the bulk of the total cellular
protein (Fig. 9A). In
contrast, a subpopulation of surface-biotinylated
CD81-
3
1 complexes from HT1080 cells was
present in the void volume (Fig. 9B), perhaps due to
association with lipid raftlike domains. However, the majority of
CD81-
3
1 complexes were retained within
the column (Fig. 9B, lanes 3-11). An
additional population of CD81 (lanes 11-14)
appeared not to be associated with other large cell surface proteins.
Due to the presence of 1% Brij 96 in these experiments, size cannot be
determined accurately. Nonetheless, the results suggest that nearly all
of the CD9 complexes and most of the CD81 complexes may be considerably smaller than 4 × 106 Da.
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Fig. 9.
Gel filtration of CD9 and CD81
complexes. A, A431 cells were surface-biotinylated and
lysed in 1% Brij 96, and the extract was fractionated by size
exclusion chromatography on Sepharose CL6B. CD9 was immunoprecipitated
from 50 µl of each fraction with mAb DU-ALL-1, and associated
proteins were revealed after nonreducing SDS-PAGE and blotting with
ExtrAvidin-HRP. The distribution of total protein (as determined in a
Bradford assay) is shown in the top panel.
B, a Brij 96 extract of surface-biotinylated HT1080 cells
(~5 × 106 cells in 500 µl of lysate) was
fractionated as in A, and 14 fractions of ~600 µl were
collected. CD81 was immunoprecipitated from 500 µl of each fraction
with mAb M38, and CD81 complexes were resolved by SDS-PAGE and
visualized with ExtrAvidin-HRP.
3
1-CD9 and
3
1-CD81 complexes (level 2). PtdIns
4-K-CD63 protein complexes are retained in 1% Brij 99 or 1% CHAPS
conditions but are disrupted in 1% Brij 96, 1% Triton X-100, or RIPA
conditions (Refs. 8, 30, and 31 and results not shown). Using
CD63-PtdIns 4-K as a prototype level 3 complex, we asked (a)
whether association preferentially occurred in low density vesicles,
(b) whether association is
cholesterol-dependent, and (c) what is the
relative size of the complex.
2 integrin, transferrin receptor) was observed in Fig.
11, especially in the dense fractions (Fig. 11, B and
C). PtdIns 4-K association with CD9 was also maintained in
both the dense and light fractions from a 1% Brij 99 gradient and from
the dense fractions of a 1% Brij 99 plus 0.2% Triton gradient (not
shown). Thus, TM4SF-PtdIns 4-K complexes remained intact, even when
completely solubilized and removed from light membrane lipid vesicle
fractions.
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Fig. 10.
Distribution of PtdIns 4-K in isopycnic
sucrose gradients. Brij 99, CHAPS, and Triton lysates of A431
cells were fractionated on sucrose gradients as shown in Fig. 2, and
then 50-µl aliquots of each fraction were used to quantitate PtdIns
4-K activity (using 20 µCi of [ -32P]ATP, for 30 min,
at 37 °C).
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Fig. 11.
CD63-PtdIns 4-K complexes occur in dense
fractions and do not depend on cholesterol. A431 cells were lysed
in 1% Brij 99 (A and B) or 1% Brij 99 plus
0.2% Triton X-100 (C), and then sucrose density gradients
were carried out as described in the legend to Fig. 2. (Note that
mixtures of detergents were preincubated overnight at 4 °C to form
mixed micelles.) Aliquots of pooled light membrane fractions
(LMF, 2-5) or dense fractions (DF,
9-12) were then immunoprecipitated using mAb to integrin
2 (A2-IIE10), CD71 (OKT9), or CD63 (6H1). Following
immunoprecipitation, immune complexes were analyzed for PtdIns 4-K
activity (using 5 µCi of [
-32P]ATP for 30 min at
37 °C). D, A431 cells were lysed in 1% Brij 99, and then
immunoprecipitations were carried out using mAbs indicated in Fig. 6.
Immune complexes on beads were then treated with buffer that either did
(+) or did not (
) contain 10 mM M
CD, prior to PtdIns
4-K analysis (using 5 µCi of [
-32P]ATP for 10 min at
room temperature). Note that the addition of 1 mM M
CD
directly to the PtdIns 4-K assay mixture did not alter the enzyme
activity (not shown).
3 integrin
complexes with M
CD did not result in loss of PtdIns 4-K activity
(Fig. 11D). Instead, the activity was elevated for unknown
reasons. As expected, the most PtdIns 4-K activity was associated with
CD63, less was associated with CD9 or
3 integrin, and
little was associated with negative control proteins E-cadherin and
2 integrin. Control experiments indicated that M
CD
itself had no effect on PtdIns 4-K activity when added directly to
PtdIns 4-K assay mixtures (not shown). Treatment of intact cells with
M
CD did not induce PtdIns 4-K shedding (not shown), probably because
PtdIns 4-K is associated with the inner leaflet of the plasma membrane.
Disruption of cholesterol-containing complexes from A431 cell lysates
by the addition of filipin (10 µg/ml) had no impact on PtdIns 4-K activity associated with CD63 or CD9 (not shown). In other experiments, A431 cell lysates (before immunoprecipitation of CD63-PtdIns 4-K complexes) or immobilized CD63-PtdIns 4-K complexes (after
immunoprecipitation) were incubated for 30 min at 37 °C. In neither
case was complex formation diminished.
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Fig. 12.
Fractionation of CD63-PtdIns 4-K
complexes by Sepharose CL6B gel filtration. A431 cells were
lysed in 1% Brij 99 (A) or 1% Brij 99 plus 0.2% Triton
X-100 (B), and then gel filtration was carried out as
described in the legend to Fig. 9. 120-µl aliquots of each fraction
were then immunoprecipitated using mAb anti-CD63 (6H1) and PtdIns 4-K
activity present in immune complexes was determined. In addition,
PtdIns 4-K activity present in 50 µl of each column fraction was
determined (using 5 µCi of [ -32P]ATP, for 20 min, at
room temperature). Values for CD63-associated PtdIns 4-K activity
should be multiplied by factors of 3 (A) and 100 (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1 with TM4SF proteins
CD9, CD81, or CD63 was observed in the dense fractions of sucrose
gradients carried out using three different detergent conditions (1%
Brij 99, 1% Brij 99 plus 0.2% Triton X-100, 1% Brij 96) and two
different cell types (A431 carcinoma and HT1080 fibrosarcoma).
Likewise, CD63 association with PtdIns 4-K was maintained in dense
fractions using multiple detergent conditions (1% Brij 99, 1% Brij 99 plus 0.2% Triton X-100). None of these associations were altered by the addition of actin or microtubule-disrupting agents (not shown). Thus, the density of these complexes did not appear to be influenced by
association with cytoskeletal proteins. Results shown here for CD9,
CD81, CD63,
3
1 and PtdIns 4-K complexes
are in striking contrast to results seen elsewhere for other TM4SF
protein and/or integrin complexes. For example, associations of
1 integrins with CD36 (37),
V
3 with CD47 (36),
L
2 with CD63 (49), and
1
integrins with CD982 were all
preferentially observed in large detergent-resistant raftlike microdomains.
3
1, CD63-
3
1, CD81-
3
1 nor CD63-PtdIns 4-K
complexes were dissociated upon cholesterol depletion with M
CD, regardless of whether added to intact cells, to lysates prior to
sucrose gradients, to lysates prior to immunoprecipitation, or to
immune complexes after immunoprecipitation. In this regard,
3
1 integrin association with TM4SF
proteins again is in contrast to
V
3
integrin association with CD47/IAP. The latter association occurred
largely within the light membrane fractions from a sucrose gradient and
was disrupted upon cholesterol depletion (36). It is not clear why
CD63-associated PtdIns 4-K activity actually increased upon M
CD
treatment. Possibly, the enzyme activity could be inhibited by
cholesterol itself or by a cholesterol-dependent associated
protein not yet identified. In this regard, TM4SF proteins associate
with at least one unidentified protein in a
cholesterol-dependent manner (e.g. Fig. 6 and
data not shown). Upon treatment of intact cells with M
CD,
CD9-
3
1 complexes were not dissociated but
were shed, consistent with previous demonstrations that M
CD may
induce shedding of intact, raft-derived vesicles from the outer leaflet of the plasma membrane (59, 64). Treatment with M
CD did not cause
shedding of PtdIns 4-K complexes, consistent with their not being
present on the plasma membrane outer leaflet.
3
1,
CD81-
3
1, and CD63-PtdIns 4-K each
migrated well into the included volume of Sepharose CL6B columns,
indicating sizes of substantially less than 4 million Da. This result
again is consistent with these complexes occurring outside of the
context of an ordered lipid microdomain. In contrast, CD63-
L
2 complexes seen elsewhere were
excluded from a Sepharose CL4B column, suggesting a size of >20
million Da (49). Our results also confirm the highly specific nature of
the TM4SF complexes. Of the total surface labeled protein, only a few
proteins were present in association with CD9 or CD81, whereas the
majority of other proteins (including
2
1
integrin, CD71, and E-cadherin) were not. Also, the selective
association of PtdIns 4-K with CD63, CD9, and
3
integrin, but not
2 integrin, CD71, or E-cadherin, is
consistent with the high degree of TM4SF-PtdIns 4-K selectivity seen
elsewhere (8, 31). Furthermore, proteins colocalizing in the light
membrane fractions of sucrose gradients (i.e. caveolin,
2 integrin) did not associate with CD9, CD81, or PtdIns
4-K simply because they were in that fraction.
3
1,
CD81-
3
1, or CD63-PtdIns 4-K complexes
were evident in the light membrane fractions of sucrose gradients,
where detergent-resistant lipid-protein microdomains are typically
found. Second, upon cholesterol depletion, the
CD9-
3
1 and
CD81-
3
1 complexes shifted out of the
light membrane fractions and into the dense fractions. This result was obtained either upon treatment of intact cells or treatment of a Brij
96 cell lysate. This dependence on cholesterol is typical of raft-type
microdomains (33, 34). Third, CD9-
3
1
complexes colocalized with the ganglioside GM1 on the surface of intact cells. Again, this is typical of raft-type microdomains (33, 34).
3
1 integrin had not been shown
previously to localize into raftlike microdomains, although results
elsewhere did suggest that
1 integrins (37, 68),
L
2 (38), and
V
3 integrins (36) may sometimes associate with raftlike microdomains. The association of integrins with gangliosides (69) is also consistent with raftlike colocalization. With
the increasing realization that integrin adhesive function may depend
on integrin lateral diffusion (70, 71), it will be interesting to
determine whether raft-type domains contribute to that process.
3
1, and TM4SF-PtdIns 4-K complexes, we have failed to coimmunoprecipitate caveolin or uPAR with
3
1 integrin or TM4SF proteins from
multiple cell types. Also, our TM4SF complexes are readily observed in
cell types (e.g. K562 erythroleukemia cells) that do not
contain caveolin or uPAR. In addition, extensive analysis of numerous
additional TM4SF-associated proteins has failed to yield any GPI-linked
proteins.3 The absence of
caveolin from our TM4SF-PtdIns 4-K complexes is consistent with a
previous demonstration that the majority of PtdIns 4-K from A431 cells
is present in noncaveolar light membrane fractions (42).
1 and
2 integrins as seen
elsewhere (37, 38) were insoluble in Triton X-100 and thus may differ from the TM4SF-
3
1 complexes analyzed here.
3
1 are found
in the same structures of the plasma membrane as GM1, while at the same
time GM1 can be a molecule highly enriched in caveolae of A431 cells (75).
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ACKNOWLEDGEMENT |
---|
We thank Dr. Robert Yauch for assistance with PtdIns 4-K assays.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM38903 and CA42368 and by Deutsche Forschungsgemeinschaft Grant Cl 163/1-1 (to C. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dana-Farber Cancer
Institute, Rm. D-1430, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3410; Fax: 617-632-2662; E-mail:
Martin_Hemler@DFCI.Harvard.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008650200
2 T. Kolesnikova, B. Mannion, and M. E. Hemler, manuscript submitted for publication.
3 C. Claas, C. S. Stipp, and M. E. Hemler, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
TM4SF, transmembrane-4 superfamily;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
CT-B, cholera toxin-B;
MCD, methyl-
-cyclodextrin;
PtdIns 4-K, phosphatidylinositol 4-kinase;
uPAR, urokinase-type plasminogen
activator receptor;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
HRP, horseradish peroxidase;
RIPA, radioimmune precipitation assay;
mAb, monoclonal antibody;
GPI, glycosylphosphatidylinositol;
GM1, Gal 1-3GalNAc 1-4Gal(3-2 NeuAc)
1-4Glc 1-1Cer.
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