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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3901-3908
CD44 Selectively Associates With Active Src Family Protein Tyrosine
Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains
of Human Peripheral Blood Lymphocytes
By
Subburaj Ilangumaran,
Anne Briol, and
Daniel C. Hoessli
From the Department of Pathology, Centre Médical Universitaire,
Geneva, Switzerland.
 |
ABSTRACT |
CD44 is the major cell surface receptor for the extracellular matrix
glycosaminoglycan hyaluronan and is implicated in a variety of
biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of
signal transduction via CD44 remains undefined, although CD44 was shown
to physically associate with intracellular protein tyrosine kinase Lck
in T lymphocytes. In the present report, we show that a significant
proportion of CD44 in human peripheral blood T lymphocytes and
endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions.
CD44-associated protein kinase activity was selectively recovered from
the low-density membrane fractions, corresponding to
glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of
the in vitro phosphorylated proteins showed that CD44 associates not
only with Lck but also with Fyn kinase in these membrane domains. Our
results suggest that cellular stimulation via CD44 may proceed through
the signaling machinery of glycosphingolipid-enriched plasma membrane
microdomains and, hence, depend on the functional integrity of such
domains.
| |
INTRODUCTION |
CD44 IS A TYPE I transmembrane
glycoprotein expressed in a variety of cell types, including
lymphocytes, macrophages, erythrocytes, fibroblasts, epithelial cells,
and endothelial cells.1 Although CD44 is encoded by a
single gene, alternative RNA splicing gives rise to a large number of
isoforms with distinct cellular distribution patterns.
Posttranslational addition of N- and O-glycans and chondroitin/heparan sulfate moieties make CD44 a highly heterogeneous molecule.
Furthermore, expression of certain defined isoforms is associated with
specific cellular behavior.2 CD44 is a major receptor for
the extracellular glycosaminoglycan hyaluronic acid (HA).3
Interaction with HA underlies a wide spectrum of CD44 functions in
embryonic morphogenesis and organogenesis, lymphocyte homing,
hematopoiesis, cellular activation, and tumor
progression.1,4-6
Signaling via CD44 has been well documented using anti-CD44 monoclonal
antibody (MoAb) as well as its natural ligand. Specific anti-CD44 MoAb
induces T lymphocytes to secrete interleukin-2 and proliferate, both
directly and in response to suboptimal doses of mitogenic anti-CD3 or
anti-CD2 pairs7-12; stimulates cytotoxic effector functions
in cytotoxic T lymphocytes (CTLs) and polymorphonulear leukocytes
(PMNs)12-14; and augments natural killer (NK)
cell cytotoxicity15-17 and macrophage proinflammatory
cytokine secretion.18 Similarly, HA binding to
CD44 stimulates T lymphocytes19,20 and
macrophages.21-24 In B cells, both anti-CD44 MoAb and HA
stimulate proliferation and differentiation into antibody-producing
cells.25 Marked differences were shown in the ability of
cell surface CD44 to bind HA and in the capacity of anti-CD44 MoAbs to
stimulate target cells.1 Recent studies have shown that HA
fragments, but not polymeric HA, activate macrophages via
CD44.23,24 HA fragments are generated by activated
leukocytes in chronic inflammatory lesions26,27 that might
lead to further recruitment of leukocytes. CD44-HA interaction
contributes directly to tumor development,28 and elevated
hyaluronidase production by tumors generates HA fragments that promote
neoangiogenesis.29
Despite the well-documented signaling capacity of CD44, the mechanism
of signal transduction via CD44 remains poorly understood. The recent
demonstration that CD44 coprecipitates Lck in human T
lymphocytes30 provides one important link in the CD44
signaling pathway. A significant proportion of CD44 resists
solubilization in nonionic detergents,31 and recent
investigations have shown that CD44 molecules lacking the cytoplasmic
tail are still detergent-insoluble, possibly because of their
association with Triton X-100 (TX-100)-insoluble lipids.31,32 This behavior is similar to that of most
glycosylphosphatidylinositol (GPI)-anchored
glycoproteins,33 which are confined to the external leaflet
of the plasma membrane. Because many GPI-anchored molecules transduce
signals34 and associate with Src family PTKs in specialized plasma membrane domains,35,36 we investigated whether CD44 also shares this property. Our results show that only the CD44 molecules present in glycosphingolipid (GSL)-rich low-density membrane
domains associate with active Lck as well as Fyn kinases.
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MATERIALS AND METHODS |
Reagents and antibodies.
TX-100 was from Merck (Darmstadt, Germany). Brij-58 (polyoxyethylene 20 cetyl ether) and horseradish peroxidase (HRP)-conjugated cholera toxin
were from Sigma Chemie (Buchs, Switzerland). Protease inhibitors
aprotinin, leupeptin, Pefabloc SC, biotin-X-NHS ester, and
octylglucoside (OTG) were from Boehringer Mannheim (Mannheim, Germany).
HRP-conjugated streptavidin and the enhanced chemiluminescence (ECL)
reagent were from Amersham (Buckinghamshire, UK). Mouse MoAb against human CD2 (MEM-65), CD44 (MEM-85), CD45 (MEM-28), CD59
(MEM-43), and major histocompatibility antigen I (MHC-I; MEM-147) were
kind gifts from Dr Vaclav Horejsi (Institute of Molecular Genetics,
Prague, Czech Republic). Rabbit polyclonal antibodies against Lck and
Fyn kinases, HRP-conjugated goat antimouse and goat antirabbit IgG and
protein A/G immunoprecipitation beads were from Santa Cruz
Biotechnology Inc (Santa Cruz, CA). Rabbit polyclonal antibody against
caveolin and antiphosphotyrosine MoAb PY20 were from Transduction Labs
(Lexington, KY). Alkaline phosphatase (AP)-conjugated goat antimouse
IgG was from PharMingen (San Diego, CA). BCA protein quantitation kit
and AP-ECL detection kit were from Bio-Rad (Richmond, CA).
Distribution of cellular proteins and GM1 ganglioside in equilibrium
density gradients.
Human peripheral blood mononuclear cells (PBMC) were isolated from
buffy coats. One hundred million PBMC or PHA blasts were washed in TKM
buffer (50 mmol/L Tris-HCl, pH 7.4, 25 mmol/L KCl, 5 mmol/L
MgCl2, and 1 mmol/L EGTA) and lysed in 0.750 mL of lysis buffer (TKM buffer containing 0.5% TX-100 or 0.5% Brij-58 and the
protease inhibitors leupeptin [1 µmol/L], aprotinin [2 µg/mL], and Pefabloc SC [2 mmol/L] and 100 µmol/L sodium orthovanadate) for
30 minutes on ice. After adding sucrose to 40%, the lysate was placed
at the bottom of a SW41 tube, overlaid with 6.0 mL of 36% sucrose
followed by 3.5 mL of 5% sucrose in TKM buffer, and centrifuged at
250,000g for 16 to 20 hours at 4°C. Eleven 1-mL fractions
(excluding the pellet) were collected from the top and stored at
 20°C. Human umbilical vein-derived endothelial cell line
ECV304 obtained from ATCC (Rockville, MD) was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Confluent cultures in 10-cm diameter Petri dishes were overlaid with 750 µL of lysis buffer containing 0.5% TX-100 or
60 mmol/L OTG, scraped, and incubated for 30 minutes on ice. Further
steps were the same as described above for PBMC. To analyze the
detergent extractability of cell surface proteins, postnuclear supernatants were removed by centrifuging the detergent lysate at full
speed in a table-top centrifuge for 5 minutes, and the nuclear pellet
was solubilized in 750 µL of TKM buffer by sonication.
The presence of various cell surface and intracellular proteins in the
density gradient fractions, total detergent extract, or solubilized
nuclear pellet was evaluated by Western blotting as described
earlier.37 Briefly, 20 µL of the samples solubilized in
6× sample buffer were electrophoresed and transferred to
nitrocellulose filters. After blocking, proteins were detected using
appropriate first and HRP-conjugated second antibodies by ECL.
Distribution of the GM1 ganglioside was analyzed by dot-immunoassay
using HRP-conjugated cholera toxin as detailed elsewhere.37
Western blot and dot-blot luminograms were quantitated by laser
scanning densitometry (Molecular Dynamics, Sunnyvale, CA).
Cell surface biotinylation and immunoprecipitation.
Confluent cultures of ECV304 cells were biotinylated with 50 µg/mL
Biotin-X-NHS ester in 5 mL of biotinylation buffer (10 mmol/L sodium
borate buffer, pH 8.9) at room temperature for 15 minutes, washed in
TKM buffer, and lysed in 0.750 mL of lysis buffer containing 0.5%
TX-100. After equilibrium gradient centrifugation, low-density
fractions (3 through 6, that correspond to the 5% to 36% sucrose
interface; Fig 1) were pooled. One
milliliter of the pool, precleared with Pansorbin (Calbiochem, San
Diego, CA), was added to protein A/G beads coated with MoAb against
CD44 or CD59. After incubation at 4°C for 2 hours, the beads were
washed in lysis buffer and proteins eluted by boiling in reducing
sample buffer were detected by Western blot using streptavidin-HRP and ECL reagent.
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| Fig 1.
Association of CD44 with low-density plasma membrane
fractions in human T lymphocytes. One hundred million human PBMC (left panel) or PHA blasts (right panel) were extracted in TKM buffer containing 0.5% TX-100, and the lysates were subjected to equilibrium gradient centrifugation as described in the Materials and Methods. Eleven 1-mL fractions were collected from the top, and 20 µL of each
fraction (2 through 11) was electrophoresed under nonreducing (CD44,
CD45, MHC-I, and CD59) or reducing (Lck and Fyn) conditions. Separated
proteins were detected by ECL-based Western blot. Fractions 3 through 6 correspond to the 5% to 36% sucrose interface. Data shown are
representative of at least three experiments. Identical distribution
profiles were obtained when 0.5% Brij-58 was used for extraction.
Fraction 1 did not contain any of the cell surface or intracellular
proteins tested.
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Cell stimulation via CD44.
Twenty-four-well Falcon microtiter plates were coated with 50 µg/mL
anti-CD44 MoAb (MEM-85) in phosphate-buffered saline (PBS) for 2 hours
at 37°C. Freshly isolated PBMC from healthy volunteers were
suspended in RPMI at 2 × 106/mL and equilibrated to
37°C, and 1 mL was added to the wells. At the indicated time
points, cells were pelleted down in a microfuge. The cell pellet and
cells adhering to the plate were lysed in 100 µL of boiling sodium
dodecyl sulfate (SDS) lysis buffer (10 mmol/L Tris pH 7.4, 1% SDS, 100 µmol/L sodium orthovanadate), pooled, and sonicated briefly. Aliquots
of the lysates were analyzed for phosphotyrosylated proteins or the
kinases by Western blot.
Immune complex kinase assays.
Twenty-five microliters of protein A/G plus beads was incubated with 5 µg of the indicated antibodies in 500 µL PBS for 30 minutes at
4°C and FCS was added to 0.5% (to minimize nonspecific background). This mixture was incubated for a further 30 minutes and
pelleted down. For immunoprecipitation from the total cell lysates, 10 ×106 PBMC were lysed in 1 mL of lysis
buffer containing 0.5% Brij-58 and the nuclei were removed by
centrifugation. One milliliter of total lysate or pooled floating
membrane fractions (3 through 6) or the bottom fractions (9 through 11)
from the equilibrium gradients of Brij-58 lysate were precleared with
Pansorbin and added to Ab-coated protein A/G beads. After incubation at
4°C for 2 to 4 hours on a rotating wheel, the beads were washed
twice in TKM-0.5% Brij-58, washed once in kinase buffer (20 mmol/L
MOPS, pH 7.4, 5 mmol/L MgCl2, 5 mmol/L MnCl2,
100 µmol/L sodium orthovanadate), and finally suspended in 25 µL of
kinase buffer. Kinase reaction was initiated by the addition of 2 µCi
of [ -32P]-ATP (5,000 Ci/mmol; Amersham) in 5 µL
kinase buffer. After incubation for 15 minutes at 30°C, the
reaction was stopped by adding 6× sample buffer and boiling.
Alternatively, SDS was added to 0.4% to disrupt the membrane complexes
and diluted with TKM-0.5% Brij-58 to 500 µL volume, and the in vitro
phosphorylated proteins were reprecipitated with antibodies against
phosphotyrosine, Lck, or Fyn. Immunoprecipitated proteins were analyzed
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
autoradiography.
| |
RESULTS |
Solubility of CD44 in non-ionic detergents has been reported to be cell
type specific.31 Whereas a significant fraction of CD44 in
fibroblasts was detergent-insoluble, lymphocyte and epithelial CD44
were completely solubilized in TX-100. In fibroblasts, only the
detergent-insoluble fraction of CD44 floated up to the low-density
fractions in equilibrium density gradients.32 However, when
we analyzed the flotation properties of different cell surface molecules with GPI or polypeptide membrane anchor in human peripheral blood T lymphocytes lysed in 0.5% TX-100, we observed that 20% to
30% of the extracted CD44 floated up to the low-density fractions at
the 5% to 36% sucrose interface (Fig 1). All CD44 was extracted under
these conditions (data not shown). Almost all of the GPI-anchored CD59
(Fig 1) and more than 60% of the cholera toxin binding GM1 ganglioside
(Fig 2A) were recovered from the floating
fractions 3 through 6, which also contained small amounts of MHC-I and
CD45 (Fig 1). Similar results were obtained with PHA blasts (Fig 1, right panel; and Fig 2A). Protein estimation by BCA method showed that
the floating low-density fractions (3 through 6) contained less than
20% of the total cellular proteins extracted, and the bulk remained at
the bottom of the gradient; the total protein profile across the
gradient is shown in Fig 2B.
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| Fig 2.
Distribution of the glycosphingolipid GM1 in equilibrium
sedimentation gradients. In (A), 10 µL of the gradient fractions shown in Fig 1 was dot-blotted onto nitrocellulose filters, blocked, and probed with HRP-conjugated cholera toxin followed by ECL detection. The total protein profile of the gradient fractions of TX-100 lysate of
PBMC is shown in (B). A similar profile obtained for PHA blasts is not
shown.
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We next examined the behavior of CD44 in the human endothelial cell
line ECV304 expressing caveolin, a protein also recovered preferentially in low-density membrane fractions.38 Here
again, CD44 was almost completely extracted by TX-100 (data not shown), yet 30% to 40% of CD44 was detected in GPI and caveolin-rich floating membrane fractions (Fig 3A, upper panel).
An identical distribution profile was obtained even after a stronger
extraction in 60 mmol/L OTG (Fig 3A, lower panel) or 2% TX-100
(data not shown), suggesting that the floating CD59, caveolin, and CD44
were still associated with buoyant membrane lipid complexes. We have
observed a similar pattern for murine GPI-linked Thy-1 molecules, which
were completely extracted by OTG while remaining floatable in
equilibrium gradients.37
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| Fig 3.
(A) Endothelial cell CD44 partitions into caveolin
containing low-density membrane fractions. ECV304 cells grown in 10-cm Petri dishes were extracted in TKM-0.5% TX-100 (upper panel) or 60 mmol/L OTG (lower panel) at 4°C for 30 minutes. The lysates were
subjected to equilibrium sedimentation, and the fractions were tested
for the distribution of CD44, CD59, or caveolin as described in Fig 1.
Caveolin was analyzed under reducing conditions. (B) CD44 and CD59 do
not interact directly in the low-density membrane fractions. Surface
biotinylated ECV304 cells were extracted with TKM-0.5% TX-100 and
ultracentrifuged in sucrose gradients, and the GPI-rich top fractions 3 through 6 were pooled. One milliliter of the pool was precleared with
Pansorbin and immunoprecipitated with protein A/G beads coated with
antibodies against CD59 or CD44. SDS-PAGE separated proteins were
detected by Western blot using streptavidin-HRP.
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On the plasma membrane, GSLs and cholesterol form specialized plasma
membrane microdomains39 that could be isolated as
detergent-resistant, low-density plasma membrane
vesicles.40,41 Our results show that a significant fraction
of lymphocyte and endothelial cell CD44 is recovered in membrane
fractions enriched in GPI-anchored receptors and GSLs, whether the
cells expressed caveolin or not. To investigate if CD44 and CD59 are
present in the same membrane complex in the floating membrane
fractions, we surface-biotinylated ECV304 cells, extracted in 0.5%
TX-100, and immunoprecipitated CD44 or CD59 from low-density membrane
fractions using the same lysis buffer for all the washing steps. CD44
antibodies immunoprecipitated a 90-kD molecule and did not
coprecipitate the 18- to 22-kD CD59 (Fig 3B); similarly, anti-CD59 did
not coprecipitate CD44, suggesting that recovery of CD44 in GPI-rich
membrane complexes does not arise from a direct interaction with CD59.
Protein tyrosine phosphorylation is an important step in transmembrane
signaling,42 and stimulation via CD44 leads to increased tyrosine phosphorylation.12,43,44 Stimulation of freshly
isolated human PBMC by immobilized anti-CD44 MoAb rapidly induced
phosphorylation of 59-, 56-, 42-, and 26-kD proteins on tyrosine
residues, which persisted up to 45 minutes after stimulation
(Fig 4A). The 59- and 56-kD
phosphotyrosylated proteins correspond to the positions of Lck and Fyn
tyrosine kinases (Fig 4B). This rapid induction of tyrosine
phosphorylation could result from activation of the Lck tyrosine kinase
reported to be physically associated with CD44,30 but the
mode of interaction between CD44 and Lck remains undefined. We observed
that, in freshly isolated PBMC, 20% to 30% of Lck and Fyn kinases was
recovered in the same floating fractions as CD44 and CD59, which was
increased to 50% to 60% in PHA blasts (Fig 1). This finding prompted
us to investigate if CD44 interacted with Lck as a result of its
association with distinct membrane domains. To this end, kinase assays
were performed on CD44 immunoprecipitated from total detergent lysates
or pooled density gradient fractions (Fig
5). Proteins at 55 to 60 and 80 to 90 kD were strongly phosphorylated
in the CD44 immunoprecipitate from total detergent extracts (Fig 5A), a
pattern closely resembling but stronger than that of CD59. These
interactions seem to be specific, because the CD45 immunoprecipitate
from total detergent extract showed phosphorylation of proteins in the
Src kinase region (albeit much less strongly than the CD44
immunoprecipitate) and of an additional protein of 35 kD (Fig 5A) that
has been shown to specifically associate with CD45.45 When
the experiment was performed on density gradient fractions, we observed
that the kinase activities associated with CD44 and CD59 were recovered exclusively from the low-density membrane complexes (Fig 5B, left panel, top). The CD59-associated kinase activities in the top fractions
were more apparent after a longer exposure (data not shown). The
CD44-associated phosphoproteins, particularly those in the 95-, 40- to
45-kD region were more prominent in the low-density membrane fractions than in total cell lysates. The phosphoprotein profile after kinase assay on CD44 immunoprecipitated from the pooled
bottom fractions 9 through 11, which contained more than half of
cellular CD44 and the kinases (Fig 1) and 80% of the cellular proteins
(Fig 2), was not significantly different from that of control
immunoprecipitations (Fig 5B, right panel, bottom). However, the
CD45-associated 35-kD phosphoprotein was recovered only from the bottom
fractions. Reprecipitation of the in vitro phosphorylated proteins
associated with CD44 from the floating membrane fractions showed that
they are tyrosine phosphorylated and correspond to the multiply
phosphorylated forms of Lck and Fyn (Fig
6). Fyn appears to be less efficiently phosphorylated than Lck,
suggesting that Lck is primarily responsible for CD44-associated
tyrosine kinase activities in T lymphocytes. The 80- to 90-kD
phosphoprotein does not appear to be CD44, because anti-CD44 did not
precipitate it (Fig 6). Tyrosine-phosphorylated proteins of similar
molecular weight have been observed in association with several
GPI-anchored proteins,35,46 raising the possibility that
they could be the putative, signal-transducing transmembrane linkers,
but their identities remain to be established.
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| Fig 4.
Stimulation of protein tyrosine phosphorylation via CD44.
Freshly isolated human PBMC were stimulated by immobilized anti-CD44 MoAb as described in the Materials and Methods. At the indicated time
points, cells were lysed and equivalent amounts of lysates were
electrophoresed under reducing conditions, blotted, and probed for
tyrosine phosphorylated proteins by PY20 followed by GAM-AP and ECL
detection (upper panel). Lane 1, unstimulated cells; lanes 2 through 6, cells plated in CD44 MoAb-coated wells, lane 7, cells plated in control
mouse IgG-coated wells (C). Equivalent protein loading in the wells was
verified by Ponceau-S staining of the blots, as well as by probing for
Lck and Fyn kinases (lower panels).
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| Fig 5.
Kinase activities associated with CD44 are localized in
low-density plasma membrane domains. (A) One milliliter of total
Brij-58 lysate from 10 ×106 PBMC was precleared with
Pansorbin and immunoprecipitated on protein A/G beads coated with
indicated antibodies or a control MoAb (against hapten TNP) and blocked
subsequently with FCS. After washing, the beads were assayed for the
associated kinase activity at 30°C for 15 minutes. The reaction was
stopped by boiling in sample buffer, and the phosphorylated proteins
separated by SDS-PAGE were detected by autoradiography. (B) After
gradient centrifugation of the Brij-58 extract as in Fig 1, the top (3 through 5) and bottom (9 through 11) fractions were pooled separately.
The bottom pool containing 80% of the cellular proteins was diluted
four times to equalize the protein concentration. Immunoprecipitation with indicated antibodies from the pooled top or bottom fractions and
kinase reaction were performed as in (A). The autoradiograms were
obtained after overnight exposure at room temperature. Similar results
were obtained with PHA blasts (not shown). Kinase activities associated
with CD44 are better preserved in whole lysates or gradient top
fractions when Brij-58 was used; experiments with TX-100-lysed samples
required a much longer exposure time, but showed an identical
phosphoprotein profile (not shown).
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| Fig 6.
CD44 is associated with Lck and Fyn kinases in the
low-density membrane domains. After kinase assay on CD44
immunoprecipitates from pooled low-density fractions as in Fig 5B, left
panel top, the immune complexes were washed in 0.5% Brij-58 lysis
buffer, dissociated by SDS, and reimmunoprecipitated using the
indicated antibodies. The immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography after 4 days of exposure. The total phosphoprotein profile associated with the primary CD44 immunoprecipitate is shown in the first lane.
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DISCUSSION |
CD44 transduces activation signals in T lymphocytes, CTLs, PMNs, NK
cells, B lymphocytes, and macrophages,7-25 but how exactly these signals are transduced across the plasma membrane remains poorly
understood. MoAb-induced CD44 cross-linking increases protein tyrosine
phosphorylation, calcium influx, and gene activation in target
cells.10,12,22,43 Interaction of CD44 with cytoskeletal networks and GTP,47 intracellular PTK Lck,30
and p185 HER2 transmembrane oncoprotein with PTK activity48
have been implicated as transmembrane signaling mechanisms. In the
present report, we show that (1) only a small proportion of all CD44
receptors associates with Lck, (2) this interaction occurs in
specialized plasma membrane microdomains, and (3) in addition to Lck,
Fyn is also recovered in association with CD44 from these membrane domains. These membrane domains, enriched in GSLs and GPI-anchored glycoproteins, also harbor a number of other signaling molecules and
thus represent privileged signaling sites in the plasma
membrane.39
GSL-rich plasma membrane rafts are conveniently isolated as floating
membrane vesicles in isopyknic density gradients.40,41 Whereas the association with buoyant membrane domains of GPI-anchored proteins arises from specific interactions of their lipid anchors with
GSLs,49 the acyl modification of CD44 at the
C-terminus47 is unlikely to associate the molecule with
low-density membrane complexes, because tailless mutants of CD44 were
still detergent-insoluble and buoyant.32 Although CD44 does
not seem to interact directly with GPI-anchored proteins (see Fig 3B),
interactions with GSLs through the carbohydrate head groups that could
engage a fraction of CD44 in such low-density membrane complexes are
not ruled out. It is interesting to note that GSLs are also potent
signal transducers50,51 as GPI-anchored glycoproteins, and
that their expression, like that of CD44, is modulated in metastatic
tumor cells.52
Ligand-mediated interactions with transmembrane signal transducing
receptors having PTK activity in their cytoplasmic domains underlie the
signaling capacity of some GPI-anchored proteins such as receptors for
ciliary neurotrophic factor (CNTFR- ) and glial-cell derived
neurotrophic factor (GDNFR-).53 Interactions with
integrins constitutes another mode of signaling via GPI-anchored proteins, eg, CD87 (uPAR) and CD16b (FcRIIIB).54,55 Most
other GPI-anchored proteins appear to signal through their association with intracellular Src family PTKs in GSL-rich plasma membrane domains.35,36,56 Fc RI, a transmembrane protein complex,
also appears to signal through its association with Lyn kinase in
similar membrane domains57 and critically depends on the
integrity of such domains for signaling.58 In addition to
PTKs, the GPI, GSLs, and caveolin-rich domains contain other signal
transduction molecules that include G proteins,38 calcium
channels, and enzymes involved in phosphoinositide
metabolism.59,60 Cross-linking is believed to induce
coalescence of these domains, initiate protein tyrosine
phosphorylation, and generate activation signals.39
Unlike the GPI-anchored glycoproteins, which are confined to the
external leaflet of the plasma membrane, CD44 traverses the membrane
bilayer. However, the cytoplasmic domain of CD44 does not seem to
contain any sequence motif that could mediate binding of Src
kinases.30 Whether the interactions of CD44 with Lck and
Fyn depend entirely on the lipid milieu of the buoyant membrane domains
or involve additional linker protein(s) is not known at present.
Nevertheless, this association raises the possibility that signaling
via CD44 can result from the aggregation of functional membrane rafts
as in the case of several GPI-anchored proteins. The observation that
HA polymers are poor agonists compared to fragmented HA23
supports this hypothesis and suggests that HA polymers might immobilize
the membrane domains and prevent their coalescence. Also, it should be
noted that F(ab)2 fragments of anti-CD44 MoAb significantly
inhibit the HA-induced proliferation of B lymphocytes,25 probably preventing domain coalescence. Considerable variations in the
stimulatory effects of HA on human T cells versus mouse T cells and B
cells versus T cells25 could arise from the differences in
the polymer composition of commercially obtained HA23 as
much as from the cell-type specificity of the responses. Moreover, some
anti-CD44 MoAbs stimulate cells, whereas others are ineffective or even
inhibitory1 and suppress inflammation.61,62
Clearly, further investigations are required to fully understand the
subtleties of signaling via CD44 in different cell types.
Recently, it has been demonstrated that induction of HA binding
capacity on T cells could result from activation-induced clustering of
CD44 followed by disulfide bond-mediated dimerization.63 Whether homodimerization is required for signaling via CD44 is not
known. Covalent dimerization involves the Cys 286 residue in the
transmembrane domain of CD4463 that is essential for binding high levels of HA.64 It is noteworthy that (1) the
transmembrane domain alone without the cytoplasmic tail is sufficient
to confer detergent insolubility and buoyancy,32 and (2)
only a small fraction of total cell surface CD44 is detergent
insoluble, buoyant,32 can form homodimers to confer
increased HA binding,63 and associates with Src family PTKs
in the low-density membrane domains (this report). We are currently
investigating whether the fraction of CD44 associated with
glycosphingolipid-rich membrane rafts is both signal competent CD44 and
contributes to high-affinity HA binding induced upon cellular
activation.
| |
FOOTNOTES |
Submitted June 30, 1997;
accepted January 12, 1998.
Supported by the Swiss National Science Foundation Grant No. 31-39 709.93 and by Swiss Cancer League Grant No. 462-2-1997. S.I. is
supported by a grant from Sir Jules Thorn Charitable Trusts.
Address reprint requests to Daniel C. Hoessli, MD,
Department of Pathology, Centre Médical Universitaire, 1 rue
Michel Servet, 1211 Geneva 4, Switzerland; e-mail:
Daniel.Hoessli{at}medecine.unige.ch.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Vaclav Horejsi for his generous gift of
antibodies.
| |
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Blood,
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[Abstract]
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A. Kosugi, S.-i. Saitoh, S. Noda, K. Yasuda, F. Hayashi, M. Ogata, and T. Hamaoka
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[Abstract]
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S. Oliferenko, K. Paiha, T. Harder, V. Gerke, C. Schwarzler, H. Schwarz, H. Beug, U. Gunthert, and L. A. Huber
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[Abstract]
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A. Trkola, C. Gordon, J. Matthews, E. Maxwell, T. Ketas, L. Czaplewski, A. E. I. Proudfoot, and J. P. Moore
The CC-Chemokine RANTES Increases the Attachment of Human Immunodeficiency Virus Type 1 to Target Cells via Glycosaminoglycans and Also Activates a Signal Transduction Pathway That Enhances Viral Infectivity
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H. Ishiwatari-Hayasaka, T. Fujimoto, T. Osawa, T. Hirama, N. Toyama-Sorimachi, and M. Miyasaka
Requirements for Signal Delivery Through CD44: Analysis Using CD44-Fas Chimeric Proteins
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U. Dianzani, M. Bragardo, A. Tosti, L. Ruggeri, I. Volpi, M. Casucci, F. Bottarel, M. J. Feito, S. Bonissoni, and A. Velardi
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S. P. Evanko, J. C. Angello, and T. N. Wight
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S. Ilangumaran, S. Arni, G. van Echten-Deckert, B. Borisch, and D. C. Hoessli
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R. Li, N. Wong, M. D. Jabali, and P. Johnson
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Abstract
Introduction
Abstract