From the Dana-Farber Cancer Institute and the
Department of Pathology, Harvard Medical School, Boston,
Massachusetts 02115 and the § Department of Pathology and
the University of Colorado Cancer Center, University of Colorado
Health Sciences Center, Denver, Colorado 80262
Received for publication, October 29, 2000
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ABSTRACT |
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CD81 and CD9, members of the transmembrane-4
superfamily (TM4SF; tetraspanins), form extensive complexes with other
TM4SF proteins, integrins, and other proteins, especially in mild
detergents. In moderately stringent Brij 96 lysis conditions, CD81 and
CD9 complexes are virtually identical to each other, but clearly
distinct from other TM4SF complexes. One of the most prominent proteins within CD81 and CD9 complexes is identified here as FPRP, the 133-kDa
prostaglandin F2 CD81, a member of the transmembrane-4 superfamily
(TM4SF),1 also called
tetraspanins, was first identified as the target of an antibody that
inhibited proliferation of a B lymphoma cell line (1). Subsequently, it
has been implicated in B cell signaling and activation (2), thymus
functions (3), T cell co-stimulation (4), myoblast fusion and myotube
maintenance (5), and neurite outgrowth (6) and as a receptor for
hepatitis C virus (7). Knockout of CD81 in mouse confirmed that it has
a role in B cell signaling and activation (8, 9) and in regulation of T
cell proliferation (10). Another tetraspanin protein, CD9, was
identified first as a lymphohemopoietic marker (11) and later was
implicated in cell motility (12), metastasis (13), heparin-binding
epidermal growth factor activity (14), neurite outgrowth (15),
myotube formation and maintenance (5), and sperm-egg binding and fusion (16). Consistent with this, knockout of mouse CD9 caused reduced fertilization due to impaired sperm-egg fusion (17, 18).
As TM4SF proteins, CD81 and CD9 possess short cytoplasmic amino and
carboxyl termini and a short cytoplasmic loop, all of which lack any
obvious signaling motifs (19-21). Instead of signaling directly, TM4SF
proteins may act as facilitators or adaptors (20) connecting a subset
of cell-surface proteins to a network described as the tetraspan web
(22). In this regard, CD81- and/or CD9-interacting proteins identified
to date include Leu-13, CD19, CD21, HLA-DR, major histocompatibility
complex I, CD4, CD8, Associations between CD81 or CD9 and other molecules may be
functionally meaningful. For example, antibodies to CD19 and CD81 both
trigger homotypic aggregation (23), whereas a CD19 chimera that no
longer associates with CD81 no longer mediates this effect (31). Also,
B cells from CD81-null mice have reduced levels of cell-surface CD19
and impaired calcium fluxes in response to CD19 cross-linking (8-10).
Other studies have shown that anti-CD81 antibodies may both stimulate
(32) and interfere with (6, 33, 34) the functions of the
CD81-associated integrins, and ectopic expression of CD9 may modulate
integrin signaling (35).
Currently, little is known about the structural basis of the proposed
TM4SF network. The large number of putative CD81- and CD9-associated
proteins raises questions about the size of these complexes and the
proximity and specificity of individual associations. In this regard,
gel permeation analysis of TM4SF complexes in CHAPS lysates suggests
that these complexes could be in excess of 20 × 106
Da (36). In addition, isopycnic sucrose gradient results suggest that
many TM4SF associations may exist in the context of large detergent-resistant membrane microdomains (37, 38). Consistent with
this, a large number of CD81 and CD9 associations are seen in
relatively mild (i.e. permissive) detergents such as CHAPS, Tween 20, Brij 58, and Brij 99 (22, 39, 40) that are generally less
able to disrupt microdomain-containing vesicles in cell lysates. The
tendency of TM4SF proteins to associate with each other raises critical
issues regarding specificity. For example, in CHAPS extracts of Raji
cells, anti-CD37, anti-CD53, anti-CD82, and anti-CD81 antibodies all
coprecipitated several major bands of identical molecular mass (27).
When transfected into Raji cells, CD9 acquired a pattern of
coprecipitating bands that was very similar to that of CD81 and CD82
(22). CD9 and CD63 immunoprecipitations from CHAPS extracts of MV3 and
MEL-FC cells appeared very similar (41), and CD53, CD63, CD81, and CD82
precipitated from Brij 58 extracts of Molt-4 cells all yielded similar
patterns of coprecipitating proteins, one of which was identified as
Resolution of TM4SF specificity and proximity issues may be facilitated
by careful choice of detergent lysis conditions. Detergents such as
CHAPS, Tween 20, Brij 58, and Brij 99 may be of limited utility because
they yield perhaps too many TM4SF-associated proteins. On the other
hand, a more stringent detergent such as Triton X-100 may be too
disruptive with respect to TM4SF protein associations. A useful
compromise may be a moderately stringent detergent such as Brij 96/97,
which yields a restricted set of CD81 and CD9 associations. For example
in 1% Brij 96, associations with Proteins so far shown to associate with CD81 or CD9 in Brij 96/97
conditions have not shown a very high stoichiometry. For example, in B
cells, <10% of CD19 associated with CD81 and CD9 (44) and only 1-2%
of CD4 associated with CD81 (45). Similarly, only a low level of
Antibodies--
The anti-integrin mAbs used in this study were
anti- Cell Culture--
HT1080 fibrosarcoma cells, NT2 teratocarcinoma
cells, and 293 cells were maintained in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum (Life Technologies, Inc.),
penicillin/streptomycin, and 2 mM glutamine. Retinoic
acid-treated NT2 cells (NT2RA cells) were obtained by treating NT2
cells with 10 µM retinoic acid for 4-5 weeks, splitting
cells 1:6 into fresh flasks, and treating them for 10-14 days with
mitotic inhibitors (56). These cells consist of a mixed population of
neuron-like and non-neuronal cell types. NT2 neuron-like cells (NT2N
cells) were purified from NT2RA cells as previously described (6).
Purified neurons were maintained in serum-free Dulbecco's modified
Eagle's medium with B27 supplements (Life Technologies, Inc.)
(57) on plates coated with Matrigel (Becton Dickinson) diluted
1:30.
Immunoprecipitation and Immunoblotting--
Cells were
biotinylated with 0.2 mg/ml sulfosuccinimidyl
6-(biotinamido)hexanoate (Pierce) in 20 mM HEPES (pH 7.5),
150 mM NaCl, and 5 mM MgCl2 (HBSM)
for 1 h at room temperature. After three rinses with HBSM, cells
were lysed by scraping into 1% Brij 96 (polyoxyethylene 10-oleyl
ether; Fluka) in HBSM with 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 10 µg/ml leupeptin. After a 1-h
extraction at 4 °C with rocking, insoluble material was removed by
centrifugation, and lysates were precleared for 1 h at 4 °C with protein G-Sepharose (Amersham Pharmacia Biotech). Specific antibodies were added along with protein G-Sepharose, and immune complexes were collected overnight at 4 °C. After rinsing four times
with lysis buffer, immune complexes were eluted by boiling in sample
buffer, resolved by SDS-PAGE, and transferred to nitrocellulose. Blots
were blocked with 3% nonfat milk in phosphate-buffered saline with
0.1% Tween 20 (PBST). After rinsing with PBST, blots were developed
with HRP-ExtrAvidin (Sigma) and diluted 1:3000 in PBST, followed by
chemiluminescence. For immunoblotting, CD81-associated proteins were
eluted from rinsed immune complexes with 1% Triton X-100 (Sigma) and
0.2% SDS in lysis buffer. Eluted proteins were separated by SDS-PAGE,
transferred to nitrocellulose, and blocked with 5% nonfat milk in PBST
or with 2% bovine serum albumin and 2% Tween 20 in phosphate-buffered
saline (for FPRP blots). Blots were developed with diluted antibodies
for Purification of CD81-associated Proteins--
~7 × 108 NT2RA cells were lysed in a total of 12 ml of 1% Brij
96 lysis buffer and precleared as described above. Anti-CD81 antibody
JS64 was added at 5 µg/ml along with 160 µl (settled volume) of
protein G-Sepharose, and CD81 complexes were collected overnight at
4 °C. After extensive rinsing with lysis buffer, CD81-associated
proteins were eluted by incubation with 1% Triton X-100 and 0.1% SDS
in HBSM with 20 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride for 10 min at 37 °C.
Eluted proteins were collected in ~250 µl with a
microcentrifuge filter unit (Costar Corp.). Using an Amido Black dye
binding assay (58), we estimated that ~36 µg of CD81-associated
proteins were recovered in the eluate. The eluate was concentrated with
a Microcon-10 microconcentrator (Amicon, Inc.) and resolved by SDS-PAGE
on an 11% minigel. CD81-associated proteins were revealed by silver staining, excised, rinsed with 50% HPLC-grade acetonitrile, and stored
at Nano Liquid Chromatography Ion Trap Tandem Mass
Spectrometry and Sequencing--
Silver-stained bands were subjected
to in-gel reduction, carboxyamidomethylation, and tryptic digestion
(Promega). Multiple peptide sequences were determined in a single run
by microcapillary reverse-phase chromatography directly coupled to a
Finnigan LCQ quadrupole ion trap mass spectrometer equipped with a
custom nanoelectrospray source. The column was packed in-house with 5 cm of C18 support into a New Objective one-piece 75-µm
inner diameter column terminating in an 8.5-µm tip. Flow rate was 190 nl/min. During chromatography, the ion trap repetitively surveyed
full-scan mass spectra over the range of m/z
300-1400, executing data-dependent scans on the three most
abundant ions in the survey scan. These scans allowed acquisition of a
high resolution (zoom) scan to determine charge state and exact mass
and tandem mass spectra for peptide sequence information. Tandem
mass spectra were acquired with a relative collision energy of 30%, an
isolation width of 2.5 Da, and recurring ions dynamically excluded.
Interpretation of the resulting tandem mass spectra of the peptides was
facilitated by programs developed in the Harvard Microchemistry
Facility and by data base correlation with the algorithm Sequest (59,
60).
Sucrose Gradients--
Brij 96 lysates of cell
surface-biotinylated NT2RA cells were prepared as described above.
Methyl- Gel Permeation Chromatography--
Cell surface-biotinylated 293 cells were lysed in 1% Brij 96 as described above. Lysate containing
~1 × 107 cell equivalents was loaded in 5%
glycerol with blue dextran and phenol red onto a 25.5 × 1.0-cm
Sepharose 6B column that had been pre-equilibrated in 1% Brij 96 in
HBSM at room temperature (Brij 96 solutions cloud upon prolonged
storage at 4 °C). 22 fractions of ~540 µl were collected,
spanning from the leading edge of the blue dextran elution to the
phenol red elution point. CD81 complexes were immunoprecipitated from
each entire fraction and analyzed by SDS-PAGE as described above.
Immunodepletion--
~4 × 107 293 cells were
surface-biotinylated and lysed in 1% Brij 96 as described above. The
lysate was divided into six equal portions to be immunodepleted four
times with either protein G-Sepharose alone or with protein G-Sepharose
plus mAb specific for CD9, CD81, CD151, CD81 and CD9 Complexes Are Distinct from Other TM4SF
Complexes--
To address the issue of TM4SF protein complex
specificity, we first compared the profile of associated proteins for
several TM4SF proteins in two different cell types. For these
experiments, we lysed cells in 1% Brij 96 (same as Brij 97) because
this detergent (e.g. in comparison with Brij 98/99, Brij 58, or CHAPS) often yields a narrower range of TM4SF-associated proteins.
After cell-surface labeling with biotin, individual TM4SF complexes
were immunoprecipitated from Brij 96 lysates. In retinoic acid-treated
NT2 cells (NT2RA cells), the profiles of CD81- and CD9-associated
proteins were virtually identical and were distinct from the profiles
of other TM4SF proteins and integrins (Fig.
1A). For example, major CD9- and CD81-associated species migrating at ~40, 45, and 70 kDa
(asterisks) were much less abundant in or completely absent
from other TM4SF complexes. Conversely, a major 36-kDa species present
in CD82 complexes was absent from CD9 and CD81 complexes. In 293 cells, CD81 and CD9 complexes were again virtually identical to each other and
contained major species at ~52 and 70 kDa (Fig. 1B, asterisks) that were much less abundant in or absent from
other TM4SF complexes. Common to all the TM4SF complexes was the
presence of species above 120 kDa that comigrated with integrins. These results provide confidence that CD81 and CD9 complexes may be substantially distinct from other TM4SF complexes and from integrin complexes.
CD81 Complexes Contain a Major Unidentified 133-kDa
Protein--
To examine more carefully the integrin-like material in
CD81 complexes, we prepared Brij 96 lysates and then fractionated CD81-associated proteins on low percentage SDS-polyacrylamide gels. In
NT2RA cells, CD81 immunoprecipitation yielded coprecipitation of a
major cell-surface biotin-labeled protein of ~133 kDa (Fig. 2A, lane 1). As
seen by
In a separate experiment, CD81 complexes from surface-biotinylated
NT2RA cells were immunoprecipitated and dissociated with 1% Triton
X-100 and 0.2% SDS, and then proteins were re-immunoprecipitated with
mAb to Identification of FPRP as a Major CD81-associated Protein--
To
identify the 133-kDa species, we prepared CD81 immune complexes from
Brij 96 lysates of NT2RA cells, and CD81-associated proteins were
eluted with 1% Triton and 0.2% SDS (see "Materials and Methods").
SDS-PAGE analysis of 6 µg of CD81-associated proteins, followed by
silver staining, again revealed a major 133-kDa species that was
apparently more abundant than any other CD81-associated protein (Fig.
3, left lane). Next, ~36
µg of CD81-associated proteins were fractionated by SDS-PAGE, and
silver-stained material above 120 kDa was excised for further analysis.
Proteins were digested in situ with trypsin, and the eluted
tryptic peptides were separated by reverse-phase liquid chromatography
and sequenced by ion trap tandem mass spectrometry. We obtained
sequence information for one peptide derived from the
To verify that FPRP associates with CD81, we analyzed CD81
immunoprecipitates by immunoblotting with an anti-FPRP polyclonal antibody. The anti-FPRP polyclonal antibody recognized a single band
from NT2RA cells (Fig. 4, lane
3) and from 293 cells (lane 9) that precisely
comigrated with the major 133-kDa band seen in total cell
surface-biotinylated CD81-associated proteins (lanes 1 and
7). The FPRP Associates Specifically with CD81 and CD9--
To determine
whether FPRP associates with other TM4SF proteins and/or integrins,
TM4SF and integrin immunoprecipitates were prepared from Brij 96 lysates and then blotted for FPRP. FPRP was detected specifically in
CD81 complexes, but not in CD151,
Because CD9 and CD81 yielded virtually identical patterns of
coprecipitating proteins in multiple cell lysates (see Fig. 1 above),
it was not unexpected that CD9 immunoprecipitates from 293 cells would
also contain FPRP (Fig. 5A). It is unlikely that CD9 is
required for CD81-FPRP association since CD81·FPRP complexes were
observed in two cell lines (NT2N and HT1080) (Fig. 5, B and C) that lack CD9 (6, 29).
CD81-FPRP Association Is Not Lipid
Raft-dependent--
Lipid rafts are membrane microdomains,
stabilized by a high content of cholesterol, sphingolipids, and
phospholipids with long, saturated fatty acyl side chains (61, 62). The
importance of cholesterol is underscored by the raft-destabilizing
effect of the cholesterol-depleting agent M
Here we analyzed CD81·CD9·FPRP complexes to determine (i) whether
they would localize to low density "light membrane" fractions of
sucrose gradients, and (ii) if they would be perturbed upon cholesterol
depletion with M
The identity of other CD81-associated proteins (e.g. the
~75-kDa protein seen in Fig. 6A) remains to be determined.
Notably, a 45-kDa protein species (Fig. 6A, upper
panel, open arrow) was no longer detected after M
For an even more rigorous test of cholesterol dependence of the
CD81·CD9·FPRP complex, we incubated cell surface-biotinylated, immunopurified CD81 complexes with increasing concentrations of M Stoichiometry of CD81-CD9-FPRP Association--
To estimate the
fraction of FPRP that is associated with CD81 and CD9, we performed
immunodepletion experiments. Brij 96 lysates of surface-biotinylated
293 cells were subjected to four rounds of immunodepletion using either
no antibody or anti-CD9, anti-CD81, anti-CD151, anti-
Immunodepletion of CD151, Different CD81 Complexes Have Distinct Sizes--
For further
analysis of CD81 complexes, we utilized gel permeation chromatography.
A cell surface-biotinylated Brij 96 extract of 293 cells was
fractionated on a Sepharose 6B column, and then CD81 complexes were
immunoprecipitated from each fraction and analyzed by SDS-PAGE. All of
the CD81 complexes eluted significantly before the approximate void
volume of the column as measured by the leading edge of dextran blue
(shown in Fig. 8A; quantified in Fig. 8B). Thus, in Brij 96 lysates, CD81 complexes exist
as discrete units of limited size (<4 × 106 Da). The
133-kDa FPRP protein eluted in a broad peak centered in fractions
6-10. These peak fractions also contained comparatively small amounts
of labeled CD9 and CD81. (Note that surface labeling of TM4SF proteins
may be blocked by TM4SF-associated molecules.) These results indicate
that most CD81·FPRP complexes also contain CD9 in 293 cells.
Whereas CD81-associated FPRP peaked in fractions 7 and 8, the bulk of
labeled CD81 peaked in fractions 11-16 (Fig. 8). Thus, we roughly
estimate that 30-50% of labeled CD81 may not be associated with FPRP.
In addition, an unknown CD81-associated 75-kDa protein (p75) peaked in
fractions 8-10, suggesting that this complex is at least partly
distinct from the CD81·FPRP complex. Also, levels of CD9 appearing in
fractions 10-13 suggest that some p75·CD9·CD81 and/or CD81·CD9
complexes are present and are distinct from CD81·FPRP complexes.
Identification of a Novel CD81 (and CD9)-associated
Protein--
Our primary goal was to identify novel components
abundantly present in CD81 protein complexes. However, it was first
necessary to address the issue of TM4SF specificity, given the tendency of TM4SF proteins to associate with each other and with so many other
proteins. In this regard, we established that CD81 and CD9 complexes
can have a composition that is quite distinct from that of other TM4SF
complexes (CD82, CD63, and CD151) and integrin complexes
(
Analysis of immunopurified CD81 complexes by cell-surface biotin
labeling or by silver staining revealed that in some cell lines, one of
the most prominent CD81-associated proteins was a potentially novel
protein of 133 kDa, distinct from integrins. By ion trap tandem mass
sequencing, this 133-kDa protein was identified definitively as FPRP.
FPRP is an Ig superfamily protein with six Ig domains that was
originally copurified with a prostaglandin F2
The CD81·FPRP complex was elevated in some cell lines (293 kidney
epithelial cells and NT2RA neuronal cells) and less abundant in others
(HT1080 fibrosarcoma cells). When FPRP was present at high levels
(e.g. in 293 cells), not only was it one of the most abundant CD81-associated proteins, but it also showed very high stoichiometry, with nearly 100% of surface-labeled FPRP associating with both CD81 and CD9. In contrast, previously reported CD81- and
CD9-associated proteins such as Specificity, Size, and Density of TM4SF Protein Complexes--
Our
secondary goal was to examine issues of TM4SF complex specificity,
size, and density. Our observations confirm that under appropriate
detergent conditions, CD81 and CD9 complexes contain unique components
not found in other TM4SF complexes. Indeed, we detected FPRP
specifically in CD81 and CD9 complexes and not in CD63, CD151,
Sucrose density gradients and gel filtration experiments revealed
further differences among CD81 complexes. For example, complexes containing major unidentified 75- and 90-kDa species were less dense
and a little smaller than CD81 complexes containing p133/FPRP. A CD81
complex with an unknown 45-kDa protein was also seen in the lower
density sucrose fractions, and this complex, unlike any of the others,
was uniquely dependent on cholesterol. Gel permeation chromatography
also revealed that a substantial pool of CD81 may exist in an
uncomplexed state on the surface of 293 cells. These results provide
evidence that TM4SF protein complexes may exist as discrete units,
rather than as large vesicular aggregates.
The physical basis for the CD81-CD9-FPRP association is unclear.
Because essentially all FPRP was associated with CD9, as well as with
CD81, and nearly all CD9 was associated with CD81, one must conclude
that essentially all FPRP was present in a CD81·CD9·FPRP complex on
293 cells. However, on HT1080 cells, CD81 associated with FPRP in the
absence of CD9, indicating that CD9 is not necessary for complex
formation. It remains to be determined whether CD9 would associate with
FPRP in the absence of CD81. Because CD9 and CD81 share more sequence
similarity with each other than with most other TM4SF proteins, one may
hypothesize that these two TM4SF proteins may share an FPRP association site.
In preliminary experiments, we found no evidence for CD81 being
directly cross-linked to FPRP.2 However, this does
not rule out a direct protein-protein interaction. The CD81 and FPRP
molecules simply may lack appropriate functional groups in proximity to
the interaction site. As an alternative, we considered the possibility
that CD81, CD9, and FPRP associate via their mutual localization to
detergent-insoluble, cholesterol-enriched membrane microdomain rafts.
Although a small fraction of CD81·CD9·FPRP complexes could indeed
colocalize with caveolin in low density fractions of isopycnic sucrose
gradients, the majority of CD81·CD9·FPRP complexes resided in
denser fractions. Furthermore, M
The majority of the CD81 complexes in sucrose gradients were clearly
more dense than typical light membrane complexes (such as those
containing caveolin) found at the 5-20% sucrose interface. However,
even after cholesterol depletion, CD81 complexes did appear to be
somewhat more buoyant than the transferrin receptor, a marker for
typical well solubilized proteins. The basis of this slight increase in
buoyancy is unclear, but one possibility is that TM4SF complexes may
contain lipids other than cholesterol such as sphingolipids and long
chain unsaturated phospholipids. Also, TM4SF proteins may display
increased buoyancy due to acylation (70, 71).
Possible Functional Relevance--
The mechanism whereby FPRP
negatively regulates prostaglandin F2
A portion of CD81·CD9·FPRP complexes appear in the light membrane
fractions of sucrose gradients, and this localization is lost upon
cholesterol depletion (e.g. see Fig. 6A). Thus,
we propose that CD81·CD9·FPRP complexes may associate with lipid
rafts. Indeed, studies elsewhere have also suggested that CD9 and CD81
may associate with lipid rafts (38, 64). In this manner,
CD81·CD9·FPRP complexes perhaps could be brought into proximity
with raft-associated signaling events, involving G proteins, Src family
kinases, and phosphoinositide metabolism.
Oocyte CD9 clearly plays a major role during fertilization (17, 18).
However, it is not yet clear whether FPRP is also present on oocytes.
If present, it would be of obvious interest to consider a potential
role for CD9·FPRP complexes during fertilization. The binding of
hepatitis C virus to CD81 is of likely relevance to liver dysfunction
and altered B lymphocyte proliferation (7). However, by Northern
blotting, FPRP appears to be absent from liver and spleen (46), so it
appears unlikely that FPRP would play a role during hepatitis C
virus binding to CD81.
In conclusion, we have identified FPRP as a major component of CD81 and
CD9 complexes in certain cell types. FPRP associates with CD81 and CD9
with high specificity and unprecedented stoichiometry. The limited
size, high density, and specificity of CD81·CD9·FPRP complexes
indicate that these exist as discrete biochemical entities, distinct
from other TM4SF complexes. The association of a portion of
CD81·CD9·FPRP complexes with lipid raft-type microdomains suggests a proximity to major signaling pathways. Together, these studies establish a novel link between TM4SF proteins and the FPRP molecule and
thus provide important new insights into the activities of CD81, CD9,
and FPRP.
receptor regulatory protein.
FPRP, a cell-surface Ig superfamily protein, associates specifically with CD81 or with CD81 and CD9, but not with integrins or other TM4SF
proteins. In contrast to other CD81- and CD9-associating proteins, FPRP
associates at very high stoichiometry, with essentially 100% of
cell-surface FPRP on 293 cells being CD81- and CD9-associated. Also,
CD81·CD9·FPRP complexes have a discrete size (<4 × 106 Da) as measured by gel permeation chromatography
and remain intact after disruption of cholesterol-rich membrane
microdomains by methyl-
-cyclodextrin. Although CD81 associated with
both
3 integrin and FPRP in 293 cells, the
3
1·CD81 and CD81·CD9·FPRP
complexes were distinct, as determined by immunoprecipitation and
immunodepletion experiments. In conclusion, our data affirm the
existence of distinct TM4SF complexes with unique compositions and
specifically characterize FPRP as the most robust, highly
stoichiometric CD81- and/or CD9-associated protein yet described.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3
1 integrin,
4
1 integrin,
6
1 integrin, other TM4SF members, CD37,
CD53, CD63, CD82, and NAG-2 (5, 14, 22-30).
4
1 integrin (30).
3
1 and
6
1 integrins are preserved, whereas other
integrin associations (seen in CHAPS, Tween 20, Brij 58, and Brij 99)
are lost (29, 42). Associations seen in Brij 96 have so far proven to
be functionally relevant. For example,
3
1·CD81 complexes contribute to neurite
outgrowth (6), and CD81·CD19 complexes modulate B cell signaling
(43).
3
1 integrin (10-20%) associated with
CD81 or CD9 in various cell lines
(29).2 In addition, in Brij
96/97 conditions, major CD81-associated cell-surface proteins have been
observed, but not yet characterized (e.g. see Refs. 29 and
44). Thus, our first goal here was to identify novel, prominent,
CD81-associated protein(s) while using relatively stringent detergent
conditions (Brij 96/97). Our second goal was to analyze novel CD81
complexes in terms of specificity, density, and estimated size. Our
results point to complexes between CD81, CD9, and FPRP (the
prostaglandin F2
receptor regulatory protein) that are
novel, robust, highly specific, and occur as discrete biochemical
entities. The FPRP molecule is a type I integral membrane protein
containing six extracellular immunoglobulin domains (46). It associates
with the prostaglandin F2
receptor and possibly other
seven-transmembrane receptors and thereby reduces receptor
ligand-binding capacity (47, 48). Aside from its associations with
seven-transmembrane receptors, no other FPRP associations had been
previously described.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2, A2-IIE10 (49); anti-
3, A3-IIF5
(50); and anti-
6, A6-BB.2 The
anti-TM4SF antibodies used were anti-CD9, DuALL (Sigma); anti-CD63, 6H1
(42); anti-CD81, M38 (51) and JS64 (52); anti-CD82, M104 (51); and
anti-CD151, 5C11 (53) and TS151r (54). The anti-transferrin receptor
mAbs used were OKT9 and HB21 (American Type Culture Collection).
Rabbit polyclonal antisera specific for
FPRP,3 the
3A
integrin cytoplasmic domain (55), and caveolin (Transduction Laboratories) were also used. Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit antibodies were from Transduction Laboratories.
3 integrin (1:1000), FPRP (1:300), caveolin
(1:5000), or transferrin receptor (1:1 mixture of OKT9 and HB21 neat
tissue culture supernatants), followed by HRP-conjugated goat
anti-rabbit or goat anti-mouse antiserum (diluted 1:4000) and chemiluminescence.
20 °C until analysis.
-cyclodextrin (M
CD) in HBSM (final concentration of 10 mM) or an equivalent volume of HBSM alone was added to
lysates containing ~2.4 × 107 cell equivalents, and
lysates were incubated for 10 min at 25 °C. Lysates were then loaded
in 45% sucrose (2 ml) over a 0.5-ml cushion of 50% sucrose and
overlaid with layers of 40% sucrose (1 ml), 20% sucrose (1 ml), and
5% sucrose (0.5 ml) prepared in HBSM without detergent. Following
centrifugation for 21 h at 45,000 rpm in a Beckman SW Ti-55 rotor
at 4 °C, 14 fractions of 360 µl were collected from the tops of
the gradients. The pellets were included in the final fraction of each
gradient. 250 µl of each fraction were diluted with 750 µl of 1%
Brij 96 in HBSM, and CD81 complexes were immunoprecipitated and
analyzed as described above. Aliquots of each fraction were also
analyzed by immunoblotting for FPRP, caveolin, and transferrin receptor.
3 integrin (all
at 10 µg/ml), or
6 integrin (1:20 dilution of
ascites). The first three rounds of immunodepletion were for 60-90 min
each; the final round was overnight at 4 °C. Triton X-100 (1% final
concentration) was added, and each sample was further divided into six
equal parts for immunoprecipitation with mAb specific for CD9, CD81,
CD151,
3 integrin, or
6 integrin or with
NeutrAvidin (Molecular Probes, Inc.) that had been coupled to Affi-Gel
10 (Bio-Rad). Samples were analyzed by SDS-PAGE and visualized with
HRP-ExtrAvidin (antibody-precipitated samples) or by immunoblotting for
FPRP (NeutrAvidin-precipitated samples).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CD81 and CD9 complexes are distinct from
other TM4SF complexes. A, ~3 × 107
NT2RA cells were cell surface-labeled with biotin and lysed in 5.5 ml
of 1% Brij 96. Equal portions of the lysate were immunoprecipitated
with antibodies to the indicated TM4SF proteins and integrins.
Complexes were resolved by SDS-PAGE on an 11% acrylamide gel,
transferred to nitrocellulose, and visualized by HRP-ExtrAvidin,
followed by chemiluminescence. Two different anti-CD151 antibodies were
used (5C11 and TS151r). Asterisks indicate bands unique to
or highly enriched in CD9 and CD81 complexes. B, ~2 × 107 293 cells were labeled with biotin and analyzed as
described for A.
3 immunoblotting (lane 2), the
~133-kDa protein (lane 1) did not comigrate with the
150-kDa
3 subunit of
3
1
integrin, the major CD81-associated integrin on NT2 cells (6).
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Fig. 2.
An unidentified 133-kDa protein in CD81
complexes from NT2RA cells. A, NT2RA and HT1080 cells
were cell surface-biotinylated and lysed in 1% Brij 96. CD81 complexes
were immunoprecipitated, eluted in 1% Triton X-100 and 0.2% SDS, and
separated by SDS-PAGE on a 7% acrylamide gel. To detect all
CD81-associated biotin-labeled proteins, blotting was carried out using
HRP-ExtrAvidin (lanes 1 and 3). Alternatively, an
anti- 3 integrin polyclonal antibody was used for
blotting (lanes 2 and 4). The locations of
3 and
1 integrin subunits and an
unidentified 133-kDa protein are indicated. B, CD81
complexes were immunoprecipitated from a Brij 96 lysate from ~8 × 107 biotin-labeled NT2RA cells. CD81-associated proteins
were eluted from the complexes in 1% Triton X-100 and 0.2% SDS. Next,
11% of the eluate was analyzed directly (lane 1), and 33%
was re-immunoprecipitated (re-ip) with an
anti-
3 integrin mAb (lane 2) or an
anti-
2 integrin mAb (lane 3). Proteins were
visualized by blotting with HRP-ExtrAvidin after SDS-PAGE.
3 (Fig. 2B, lane 2) or
2 (lane 3). Again, the relatively small
amount of re-precipitated
3
1
(lane 2) appeared quite distinct from the 133-kDa protein
co-immunoprecipitated with CD81 (lane 1). As
expected, no
2
1 integrin could be
detected in association with CD81 in a Brij 96 lysate (lane
3). In contrast to NT2RA cells, HT1080 cells yielded
CD81-coprecipitating material that resolved into bands consistent with
and
integrin subunits (Fig. 2A, lane 3).
Immunoblotting of the
3 integrin subunit (lane
4) confirmed that the upper band seen in lane 3 indeed
is the
3 integrin subunit. In conclusion, NT2RA cells,
but not HT1080 cells, possess a major CD81-associated protein of ~133
kDa that is not an integrin.
1
integrin subunit and eight peptides derived from FPRP, a cell-surface
Ig superfamily protein (46) (Table I).
The reported molecular mass of FPRP (133-135 kDa) is fully consistent
with our protein identification.
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Fig. 3.
Comparison of silver-stained and cell
surface-biotinylated CD81 complexes. ~6 µg of CD81-associated
proteins prepared from ~1 × 108 NT2RA cells were
fractionated by SDS-PAGE and visualized by silver staining. For
comparison, cell surface-biotinylated CD81-associated proteins prepared
from ~1.5 × 107 NT2RA cells were analyzed by
blotting with HRP-ExtrAvidin. The location of the major 133-kDa
CD81-associated protein is indicated.
Peptide sequences of CD81-associated proteins
3 integrin was present at a much
lower level in CD81 complexes from NT2RA and 293 cells and migrated
above FPRP (lanes 2 and 8), in agreement with
Fig. 2. Again in agreement with Fig. 2, CD81 complexes from HT1080
cells contained minimal FPRP (lane 6), whereas
3
1 integrin was abundant (lanes
4 and 5). In conclusion, the major 133-kDa
CD81-associated protein detected by cell-surface biotinylation in two
out of three cell lines is confirmed to be FPRP.
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Fig. 4.
FPRP is a major CD81-associated protein.
NT2RA cells (lanes 1-3), HT1080 cells (lanes
4-6), and 293 cells (lanes 7-9) were cell
surface-biotinylated and lysed in 1% Brij 96. CD81 complexes were
immunoprecipitated, and CD81-associated proteins were eluted in 1%
Triton X-100 and 0.2% SDS and separated by SDS-PAGE. Eluate equivalent
to ~2.5 × 106 cells was analyzed by blotting with
HRP-ExtrAvidin (lanes 1, 4, and 7),
anti- 3 integrin polyclonal antibody (lanes 2,
5, and 8), or anti-FPRP polyclonal antibody
(lanes 3, 6, and 9).
2, or
3
complexes from 293, NT2N, and HT1080 cells (Fig.
5, A-C). FPRP was also not
present in CD63 complexes (from 293 and HT1080 cells) and was not seen
in
6 complexes from NT2N cells. Results showing FPRP
present in CD81 immunoprecipitates from HT1080 cells (Fig.
5C) do not contradict results in Fig. 4 (lane 6)
because a much longer immunoblot exposure time was used in Fig.
5C.
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Fig. 5.
FPRP associates specifically with CD81 and
CD9. A, a Brij 96 lysate prepared from 293 cells was
immunoprecipitated (Ip) with mAbs against the indicated
TM4SF proteins and integrin subunits. Immunoprecipitates were eluted
with 1% Triton X-100 and 0.2% SDS, and eluates equivalent to
~3 × 106 cells were analyzed by immunoblotting for
FPRP. Independent experiments confirmed that antibodies used for
immunoprecipitation could recognize abundant CD9, CD63, CD151,
2, and
3 from 293 cells (e.g.
see Fig. 7 and not shown). B, a Brij 96 lysate of NT2N cells
was analyzed as described for A (~2 × 106 cell equivalents/lane). C, a Brij 96 lysate
of HT1080 cells was analyzed as described for A (~3 × 106 cell equivalents/lane). The antibodies utilized each
immunoprecipitated their respective antigens from NT2N and HT1080
cells, as seen in Fig. 1.
CD (63). Many
raft-localized molecules are resistant to extraction with cold nonionic
detergents, and the high ratio of lipid to protein causes the
segregation of rafts into the low density fractions (containing between
5 and 25% sucrose) of isopycnic sucrose gradients. Because TM4SF proteins (38, 64) and integrins (65-67) may sometimes localize into
lipid raft-like domains (61, 62), we considered that CD81·CD9·FPRP
complexes may occur in, or perhaps even depend on, raft-like domains.
CD. Untreated Brij 96 lysate was fractionated on a
sucrose gradient, and CD81 complexes were then immunoprecipitated from
each fraction. As shown in Fig.
6A (upper panel),
CD81, CD9, and the 133-kDa FPRP protein were broadly distributed, with
the majority of the material in fractions 4-10 (20-45% sucrose) and
a small percentage of each protein in fractions 1-3 at the top of the
gradient. Upon addition of M
CD to the lysate prior to
centrifugation, CD81, CD9, and the 133-kDa FPRP protein were depleted
from the upper fractions of the gradient, but the complexes remained
intact in the denser fractions (lower panel). Immunoblotting of CD81 immunoprecipitates with anti-FPRP polyclonal antibody confirmed
that FPRP remained associated with CD81 in either the presence or
absence of M
CD (Fig. 6B, upper and lower
panels). Unfortunately, the limited sensitivity of the anti-FPRP
polyclonal antibody precluded detection of FPRP weakly present in
fractions 1-3. Nonetheless, a shift in CD81-associated FPRP toward the
denser fractions such as seen in Fig. 6A was confirmed in
Fig. 6B.
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Fig. 6.
Effect of M CD on
CD81 complexes. A, a Brij 96 lysate was prepared from
~4.8 × 107 cell surface-biotinylated NT2RA cells.
The lysate was divided in half and left untreated (control)
or treated with 10 mM M
CD for 10 min at room temperature
(M
CD). Lysates were loaded in 2 ml of 45%
sucrose over a 0.5-ml 50% sucrose cushion and overlaid with 40% (1 ml), 20% (1 ml), and 5% sucrose (0.5 ml) prepared without detergent.
After centrifugation to equilibrium, 14 fractions of 360 µl were
collected from the top of the gradient; the pellet was included in the
final fraction. 250 µl of each fraction were used for
immunoprecipitation with an anti-CD81 mAb, and CD81 complexes were
analyzed by SDS-PAGE and blotting with HRP-ExtrAvidin. The locations of
CD9, CD81, and the major 133-kDa CD81-associated protein are indicated
by closed arrows. The open arrow indicates an
unknown 45-kDa species. B, a Brij 96 lysate prepared from
~4.8 × 107 unlabeled NT2RA cells was treated and
fractionated on sucrose gradients as described for A. CD81
complexes were immunoprecipitated from 250 µl of each fraction, and
CD81-associated proteins were eluted from the complexes with 1% Triton
X-100 and 0.2% SDS and then analyzed by immunoblotting for FPRP.
C, 20 µl of each fraction from the gradients in
B were analyzed by immunoblotting for caveolin.
D, 20 µl of each fraction from the gradients in
B were analyzed by immunoblotting for the transferrin
receptor (TfR). All gradients in A-D were
centrifuged simultaneously. E, ~3 × 107
cell surface-biotinylated NT2RA cells were lysed in 1% Brij 96, and
CD81 complexes were immunoprecipitated. CD81 complexes bound to protein
G-Sepharose were divided into equal portions and extracted with the
indicated concentrations of M
CD for 15 min at 37 °C. Complexes
remaining after extraction were eluted by boiling in SDS-PAGE sample
buffer, and 20% of each sample was analyzed by blotting with
HRP-ExtrAvidin (upper panel). The remaining 80% was
analyzed by immunoblotting for FPRP (lower panel). The
locations of CD9, CD81, and p133 are indicated by closed
arrows, and the location of a 45-kDa band removed by M
CD
treatment is indicated by the open arrow.
CD
treatment (lower panel), suggesting that at least one
protein may indeed require cholesterol for CD81 association. Caveolin,
a positive control marker of cholesterol-enriched light membranes,
localized primarily to the top four sucrose gradient fractions (Fig.
6C, upper panel). As expected, M
CD treatment substantially shifted caveolin away from the low density fractions, such that most moved to the bottom of the gradient (fraction 14), whereas some remained in fractions 3-5 (lower panel). This
caveolin was largely non-overlapping with CD81, CD9, and FPRP. The
transferrin receptor, a non-raft marker, localized somewhat below the
majority of the CD81 complexes in the sucrose gradient (predominantly
in fractions 6-12) and was largely unaffected by M
CD treatment
(Fig. 6D).
CD
prior to analysis by SDS-PAGE. As shown in Fig. 6E
(upper panel), even 100 mM M
CD failed to
disrupt the complex. Only the 45-kDa species noted in Fig.
6A was eliminated by M
CD treatment (open
arrow). Immunoblotting of an aliquot of each sample for FPRP
confirmed that M
CD had no effect on the amount of FPRP associated with CD81 (lower panel). In conclusion, the
CD81·CD9·FPRP complex may partially associate with a raft-like
microdomain distinct from caveolin-containing microdomains, but
CD81-CD9-FPRP association is not dependent on raft localization.
3
integrin, or anti-
6 integrin mAb. Triton X-100 was then
added to each sample to dissociate complexes, and the remaining amount
of FPRP, CD9, CD81, CD151,
3 integrin, and
6 integrin in each depleted sample was determined. As
shown in Fig. 7 (upper panel),
immunodepletion of either CD9 or CD81 removed essentially all
detectable FPRP. Control depletion experiments (second and
third panels) confirmed that the immunodepletion protocol had removed nearly all CD9 and CD81, respectively. Interestingly, CD81
immunodepletion removed much of the CD9 in the lysate, but CD9
immunodepletion had a minimal effect on the level of CD81. Thus, most
CD9 must be associated with CD81, whereas CD81 is in substantial excess
of CD9.
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Fig. 7.
Highly stoichiometric association of FPRP
with CD81 and CD9. ~4 × 107 293 cells were
surface-labeled with biotin and extracted with 1% Brij 96. The extract
was divided into equal portions and depleted with protein G-Sepharose
alone or with protein G plus mAb specific for CD9, CD81, CD151,
3 integrin, or
6 integrin. After four
rounds of immunodepletion, Triton X-100 was added to 1%, and each
portion was further divided into six aliquots. One aliquot was
precipitated with NeutrAvidin-agarose, followed by blotting with an
anti-FPRP polyclonal antibody. (Note that our anti-FPRP polyclonal
antibody was not suitable for immunoprecipitations.) The remaining
aliquots were immunoprecipitated with mAb specific for CD9, CD81,
3 integrin, CD151, or
6 integrin,
followed by blotting with HRP-ExtrAvidin.
3 integrin, or
6 integrin removed little if any surface-labeled FPRP
from the 293 cell lysate. In contrast, CD151 immunodepletion removed a
significant amount of the
3 integrin, consistent with
the high stoichiometry of the
3·CD151 complex observed
previously (53). CD151 immunodepletion also removed as much
6 integrin as
6 immunodepletion itself, indicating that the
6·CD151 complex is also highly
stoichiometric in 293 cells. Together, these results suggest that
specific TM4SF protein complexes (e.g. CD81·CD9·FPRP,
3·CD151, and
6·CD151) each occur at
high stoichiometry (at least with respect to CD9, FPRP,
3, and
6), but fail to interact
substantially with each other in Brij 96 lysates.
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Fig. 8.
Gel permeation analysis of CD81
complexes. A, a Brij 96 lysate from ~1 × 107 surface-biotinylated 293 cells was fractionated on a
Sepharose 6B column (25.0 × 1.0 cm) equilibrated in 1% Brij 96. 22 fractions of ~540 µl were collected from the leading edge of
blue dextran (approximating V0) to phenol red
(indicating the small molecule elution point). CD81 complexes were
immunoprecipitated from each fraction and analyzed by SDS-PAGE,
followed by blotting with HRP-ExtrAvidin. The locations of CD9, CD81,
and p133/FPRP are indicated. B, the elution profile in
A was quantified by densitometry using the program Scion
Image Version 1.62. The densities of CD9, CD81, and p133, expressed in
arbitrary (arb) units, are plotted versus
fraction number.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3,
6, and
2). A key to
these experiments was the use of 1% Brij 96/97 detergent. Less
stringent detergents such as CHAPS, Brij 58, and Brij 98/99 tend to
yield more extensive TM4SF protein complexes, in which the components
associated with several distinct TM4SF proteins appear to be identical.
In contrast, the more hydrophobic nature of Brij 96/97 yields a more
restrictive pattern of TM4SF-associated proteins (see the Introduction).
-binding
fraction (46, 68). FPRP possesses no prostaglandin F2
-binding activity of its own and, in fact,
down-regulates prostaglandin F2
binding to COS cells
when cotransfected with the prostaglandin F2
receptor (47).
3
1
integrin, CD19, and CD4 showed a much lower stoichiometry
(i.e. 1-20%; see the Introduction). In previous cases in
which high stoichiometry interactions of various proteins with CD9 or
CD81 have been observed, these were seen using less stringent
detergents such as CHAPS, Brij 99, and Brij 35 (22, 64, 69). The only
other TM4SF complexes so far reported to approach 80-100%
stoichiometry in relatively restrictive detergent conditions are the
3
1·CD151 and
6
1·CD151 complexes (Refs. 53 and 54;
see also Fig. 7).
2 integrin,
3 integrin, and
6 integrin complexes. Although both FPRP and
3 integrin associated with CD81 in 293 cells, FPRP was
not detected in
3 immunoprecipitates, and
3 immunodepletion removed no FPRP from 293 cell lysates.
Furthermore,
3
1 association with CD81 and
CD9 could be readily observed in HT1080 cells (29), which have only low
levels of FPRP; and conversely, CD81·CD9·FPRP complexes were
abundant in 293 lysates, which have relatively low levels of
3
1. When 293 cells were made to
overexpress
3
1 integrin, the amount of
FPRP associated with CD81 was not decreased.2 Thus,
3
1·CD81 (or
3
1·CD9) complexes are distinct from
CD81·CD9·FPRP complexes.
CD disruption of
cholesterol-dependent microdomains failed to dissociate
CD81·CD9·FPRP complexes, even though it was sufficient to displace
caveolin and a subset of CD81·CD9·FPRP complexes from low density
fractions. In fact, all CD81-associated proteins remained present, with
the exception of a weakly biotinylated 45-kDa species (mentioned
above). As seen here for CD81·CD9·FPRP complexes, we have found
elsewhere that
3
1·CD9 and
3
1·CD81 complexes may localize to
cholesterol-rich raft-type microdomains, but appear not to require
cholesterol or raft localization to remain intact (64).
binding is
unclear, but deletion mutagenesis suggested that this activity may map
largely to the transmembrane and cytoplasmic regions of FPRP (47). The
membrane-proximal Ig domain of FPRP contains a hydrophobic face that is
proposed to interact with the membrane or another protein. It is not
yet clear whether this hydrophobic face might interact with CD81 or
CD9. The cytoplasmic domain of FPRP contains a potential protein kinase
C phosphorylation site as well as a potential
calmodulin-dependent protein kinase phosphorylation site.
These are of potential relevance to CD81-mediated signaling events
because of the association of protein kinase C with TM4SF
complexes4 and the ability of
anti-CD81 antibodies to trigger calcium fluxes under certain conditions
(23). In addition to the prostaglandin F2
receptor, FPRP
may regulate the function of other G protein-coupled receptors,
including the
-adrenergic receptor (48), suggesting that FPRP may
associate with a range of seven-transmembrane G protein-coupled
receptors. It will be of interest to determine whether G
protein-coupled receptors can also be detected in CD81 or CD9 complexes.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge W. S. Lane, R. Robinson, and K. Pierce (Harvard Microchemistry Facility) for expertise in HPLC, mass spectrometry, and peptide sequencing.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM38903 (to M. E. H.).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 Inst., 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, November 21, 2000, DOI 10.1074/jbc.M009859200
2 C. S. Stipp and M. E. Hemler, unpublished data.
3 D. Orlicky, unpublished data.
4 X. A. Zhang and M. E. Hemler, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
TM4SF, transmembrane-4 superfamily;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;
mAb, monoclonal antibody;
HRP, horseradish peroxidase;
PAGE, polyacrylamide
gel electrophoresis;
HPLC, high pressure liquid chromatography;
MCD, methyl-
-cyclodextrin;
FPRP, prostaglandin F2
receptor regulatory protein.
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