Background
The CD98 (4F2, FRP-1) molecule, a cell surface
disulfide-linked heterodimer, was originally described as a
T cell activation antigen [ 1 ] , and later was shown to
provide a co-stimulatory signal for CD3-mediated T-cell
activation [ 2 ] , independent of CD28/CD80/CD86
interaction [ 3 ] . In other studies, triggering of human
monocyte CD98 could suppress T cell proliferation [ 4 ] ,
or promote homotypic cell aggregation of monocytes [ 5 ] .
Also, CD98 may be a target antigen for natural killer cells
[ 6 ] , may mediate fusion of blood monocytes leading to
osteoclast formation [ 7 8 ] , and may modulate
hematopoietic cell survival and differentiation [ 9 ] .
The CD98 molecule is also widely expressed on rapidly
growing non-hematopoietic cells, where it may modulate
oncogenic transformation [ 10 11 ] , metal ion transport [
12 13 ] , cell fusion [ 14 15 ] , and amino acid transport
[ 16 17 18 19 ] . CD98 is also expressed on normal
proliferating tissue such as the basal layer of squamous
epithelia [ 20 ] and on cells having secretion or transport
functions [ 21 ] . Understanding of the role of CD98 in
amino acid transport has been greatly enhanced with the
recent cloning and characterization of multiple CD98 light
chains. Distinct CD98 light chains appear to mediate
distinct amino acid transport activities (for review see [
22 23 ] ).
Several lines of evidence now suggest that CD98 may
modulate the functions of integrins. For example, CD98 and
β1 integrins may act together during T-cell co-stimulation
[ 24 ] . A genetic screen revealed that CD98 may indirectly
influence integrin affinity for ligand [ 25 ] . Also,
anti-CD98 mAb appeared to modulate integrin-dependent
adhesion in two separate studies [ 25 26 ] . In addition,
antibodies to both CD98 and α3β1 integrin promoted cell
fusion [ 27 ] , and antibodies to β1 and β2 integrins
blocked monocyte cell-cell fusion and aggregation functions
induced by an anti-CD98 mAb [ 5 ] . Finally, like CD98, the
α3β1 integrin has been implicated in amino acid transport [
28 ] .
The biochemical mechanism by which CD98 and integrins
functionally interact remains unclear. The CD98 molecule
associates specifically with immobilized β1 integrin
cytoplasmic tail fusion proteins under very mild detergent
conditions (0.05% Triton X-100) [ 29 ] , but CD98 has not
yet been shown to interact physically with intact β1
integrins. We began the present study by searching for
integrin-associated proteins in an unbiased manner (i.e. we
were not specifically looking for CD98). We utilized a
monoclonal antibody screening method involving
co-immunoprecipitation of integrins under non-stringent
detergent conditions [ 30 31 ] . Using this approach, we
have previously discovered specific associations between
particular β1 integrins and other cell surface molecules
including CD147/EMMPRIN [ 31 ] , and various tetraspanin
(transmembrane-4 superfamily) proteins [ 30 32 33 ] . In
this report we demonstrate for the first time that CD98
constitutively and specifically associates with intact β1
integrin heterodimers. Specific CD98-integrin interaction
occurs in the context of low density protein-lipid
microdomains, possibly resembling lipid rafts [ 34 35 ] .
Several important signaling molecules, including activated
TCR, LAT and LCK are present in organized raft-type
microdomains, and integrity of these microdomains is
necessary for T cell receptor signal transduction [ 36 37 ]
. Our present discovery that β1 integrins specifically
associate with CD98 in lipid microdomains helps to explain
the functional connections between β1 integrins and CD98
during T cell costimulation, monocyte fusion, and
elsewhere.
Results and Discussion
Identification of CD98 as a β1 integrin-associated
protein
Mice were immunized with MTSV1-7 human mammary
epithelial cells, and approximately 600 hybridomas were
generated. Upon monoclonal antibody screening, 16 were
identified that co-immunoprecipitated integrins without
recognizing integrins directly. Of these, one recognized
CD63 as reported previously [ 30 ] , and three (including
mAb 6B12) recognized a disulfide-linked cell surface
heterodimer consisting of an 85 kD heavy chain and a 35
kD light chain.
From I 125surface-labeled HT1080 cells, mAb 6B12
immunoprecipitated a heterodimer of 85 and 35 kD under
reducing conditions (Fig. 1, lane a, solid arrows) and a
single protein band of 120 kD under non-reducing
conditions (not shown) but no other protein bands when
lysates were prepared in 1% Triton XI 00 detergent.
However, in the less stringent Brij 58 detergent, mAb
6B12 co-immunoprecipitated an integrin-like protein band
of 130 kD (Fig. 1, lane b, dotted arrow). Similar results
were noted when CHAPS or Brij 99 detergents were used
(not shown).
Because the proteins recognized by 6B12 resembled the
disulfide-linked 85 and 35 kD subunits of CD98 [ 38 39 ]
, they were compared to authentic CD98 as precipitated
using mAb 4F2. Both mAb 4F2 and mAb 6B12 precipitated
identical patterns of cell surface proteins, as seen by
reduced SDS-PAGE (Fig. 1A, lanes c,d). Furthermore,
preincubation of cell lysates with mAb 4F2 resulted in
loss of the antigen recognized by mAb 6B12 (not shown).
Upon addition of 10 mM DTT to cell lysates, mAb 6B12
immunoprecipitated only a 85 kD protein band, consistent
with specific recognition of the CD98 heavy chain. Flow
cytometry of CHO cells transfected with CD98 heavy chain
cDNA confirmed that the CD98 heavy chain is the 6B12
antigen (Fig. 1B). Also, by flow cytometry the 6B12
antigen was present at high levels on all human cell
lines tested (more than 20) as expected for CD98.
Specificity of CD98-integrin association
To identify the integrin-like 130 kD protein(s)
co-precipitating with CD98, immune complexes prepared
from HT1080 cells were dissociated and reprecipitated
with mAb against various cell surface molecules. From
CD98 immunoprecipitates we recovered α3β1 integrin, but
not other cell surface molecules, including the LDL
receptor, HLA class I, the metalloprotease regulator
EMMPRIN (CD 147), CD71, or CD 109 (Fig. 2A). Several
other β1 integrins that are abundant on HT1080 cells
(α3β1, α2β1, α5β1, and α6β1) also could be
re-precipitated from CD98 immunoprecipitates (Fig. 2C).
Using the same experimental conditions, we were unable to
detect α4β1 in CD98 immunoprecitates prepared from the
rhabdomyosarcoma cell line RD, the lymphoblastoid cell
line Molt4, or K562 cells transfected with α4 integrin
(not shown). However, we did observe
co-immunoprecipitation of CD98 with α4β1 integrin in the
presence of 2 mM manganese (not shown). The precise role
of manganese is unclear. Incubation with anti-β1
'activating antibody' TS2/16 (not shown) did not mimic
the effects of manganese on α4β1-CD98 association. Thus,
although manganese is well known to "activate" β1
integrins [ 60 ] , this activation may not be relevant to
α4β1-CD98 association. Comparison of material isolated
from CD98 immunoprecipitates with total amounts of each
integrin on the cell surface indicates that approximately
5-10% of each integrin is associated with CD98 (Fig. 2C,
compare lanes a-d with e-h). In a reciprocal experiment,
CD98 heavy chain was re-precipitated from an α3β1
integrin immunoprecipitate (Fig. 2B, lane a) at a level
comparable to re-precipitated CD81 (lane b), a molecule
shown previously to associate with α3β1 integrins [ 32 ]
. Results in Fig. 2Aand 2Bindicate that CD98-integrin
complexes are distinct from previously described
integrin-CD81 [ 32 40 ] and integrin-CD147/EMMPRIN [ 31 ]
complexes.
Because not all β1 integrins associated constitutively
with CD98 (in the absence of manganese), the α chain must
help regulate specificity of CD98 association. Notably,
α3 with a truncated cytoplasmic tail, and wild-type α3
associated equally well with CD98 (Fig. 2D, lanes c,d).
Thus, specificity for constitutive association with CD98
may reside in integrin α chain extracellular or
transmembrane domains. To check whether CD98 is a ligand
for α3β1 integrin, we used K562 cells transfected with
wild type α3, or α3 mutants deficient in adhesion to α3
ligands laminin 5 (W220A) and invasin (Y218A) [ 41 ] .
All of these α3 proteins constructs associated with CD98
(Fig. 2E). Notably, 2 mM EDTA had no inhibitory effect on
constitutive integrin-CD98 associations (not shown).
Thus, CD98 itself does not appear to be acting as an
integrin ligand. We were unable to demonstrate any
changes in the adhesive functions of CHO cells upon
CD98-transfection. However, functional effects of
transfected human CD98 could be overshadowed by an excess
of endogenous hamster CD98.
Next we asked whether clustering of CD98 would induce
selective clustering of β1 integrins. On the surface of
RD-A3 cells, CD98 was clustered by incubation with
anti-CD98 mAb 4F2, followed by incubation with rabbit
anti-mouse antibody. Subsequent incubation with anti-α3
mAb directly conjugated to Alexa-488 revealed that α3β1
was highly clustered (Fig. 3C), unless pre-clustering of
CD98 was omitted (Fig. 3A). In contrast, staining with
anti-α4-Alexa-488 mAb revealed a diffuse pattern of
staining, regardless of CD98 clustering (Fig. 3B, 3D). If
no Alexa-488-conjugated antibodies were added, no
staining was observed (not shown). Immunofluorescent
staining of both α3β1 and CD98 showed that they were
distributed similarly on the cell surface of HT1080 and
RD-A3 cells and partially co-localized (not shown).
CD98 mutation and sucrose density gradient
localization
The CD98 heavy chain contains two extracellular
cysteines. Each of these cysteines was mutated to serine,
and the effects on β1 integrin association were
evaluated. Stably expressed human CD98 heavy chain C330S
mutant retained association with endogenous murine β1 in
NIH 3T3 cells, comparable to that seen for wild type CD98
heavy chain. However, the C109S mutant showed no
detectable β1 association (Fig. 4A, right panel). Murine
β1 (Fig. 4A, left panel), and mutant and wild type forms
of CD98 (see legend) were expressed at comparable levels
in each of the transfectants. Notably, association of
CD98 heavy chain with CD98 light chain was maintained for
the C330S mutant, but was lost for the C109S mutant (not
shown), in agreement with previously published results
suggesting that Cysteine 109 is required for disulfide
linkage to the light chain [ 42 ] . These results suggest
that the CD98 light chain might be necessary for
CD98-integrin association. One possibility is that the
light chain is more proximal to the integrin. When
suitable anti-light chain reagents become available, this
could be directly tested in covalent crosslinking
experiments. In this regard, we have thus far been unable
to crosslink CD98 heavy chain directly to β1 integrin,
using either membrane-permeable or membrane-impermeable
crosslinkers (not shown).
Another possibility is that the CD98 light chain may
target the CD98 heterodimer to the specific membrane
domain where interactions with β1 integrins may be
localized. To test the latter hypothesis, we compared the
total β1 integrin distribution with the distribution of
the fraction of β1 integrins complexed with CD98 on HT
1080 cells (Fig. 4B). Although β1 integrins were
predominantly present in the dense membrane fraction on a
sucrose gradient, the subset of β1 integrins complexed
with CD98 was localized to the 'light' membrane
fractions. These fractions typically contain caveolae and
'membrane rafts' thought to be enriched in active
signaling complexes [ 34 35 43 ] . The fraction of the β1
integrins detected in the light membrane fractions
represents no more than 5% of total β1 integrins (based
on the densitometric analysis, not shown). Though
caveolin is present in the same fractions as
CD98-integrin complexes, it does not co-immunoprecipitate
with CD98 under these conditions (not shown) and thus is
not part of integrin-CD98 complexes. Similarly,
GPI-linked proteins such as CD 109, TM4SF proteins such
as CD81, and other transmembrane proteins such as
CD147/EMMPRIN can be found at substantial levels in light
membrane fractions [ 44 45 ] , and our unpublished
results), and these also did not co-immunoprecipitate
with CD98 (Fig. 2A, 2B). Thus, CD98 association with
integrin has another level of specificity beyond simply
co-localizing in light membrane fractions.
Finally, to see whether the CD98 light chain might
help to target the CD98 heavy chain to the light membrane
fractions, we looked at the distribution of the wild type
CD98 and CD98 mutants in the sucrose gradients (Fig.
4C,D). The majority of wild type CD98 heavy chain and
control mutant (C330S) are localized to the light
membrane fraction (55-60%, Fig. 4D), whereas the C109S
mutant is partially shifted to the dense fractions (40%
in the light fraction, 50% in the dense fractions, Fig.
4D). These results are consistent with the CD98 light
chain targeting the CD98 heavy chain to light membrane
fractions. It is perhaps not surprising to observe ~40%
of the C109S heavy chain mutant remaining in the light
membrane fraction (Fig. 4C,D), considering that the CD98
heterodimer retains partial association and amino acid
transport function even in the absence of a disulfide
linkage of the subunits [ 42 ] . Notably, transformation
of BALB 3T3 cells caused by over-expression of CD98 heavy
chain required its association with the light chain, and
a missense mutation in C109 eliminated its transforming
activity [ 10 ] .
The CD98 protein was recently shown to associate
specifically with integrin β chain cytoplasmic tail
fusion proteins [ 29 ] in 0.05% Triton X-100 detergent
conditions. One may speculate that the use of 0.05%
Triton X-100 may have allowed maintenance of CD98
microdomains, needed for β1 interaction. In this regard,
in 0.05% Triton X-100 lysates, many transmembrane
proteins remain in the light membrane fractions of a
sucrose gradient [ 46 47 ] . Although the role of the
CD98 light chain was not addressed in the earlier study [
29 ] , we assume that it may have played a key role.
Furthermore, CD98 interaction with recombinant β chain
cytoplasmic tail fusion proteins, in the absence of the α
chain [ 29 ] , is consistent with our observation that
the α chain cytoplasmic tail is not needed for
integrin-CD98 association. On the other hand, we observed
CD98 interaction with several β1 integrins, but not α4β1.
Thus for intact integrins in the context of mammalian
cells, the integrin β chain apparently does contribute to
the specificity of CD98 interaction. The α chain might
provide a level of specificity by changing the
conformation of the β chain and/or by providing an
additional site of interaction with the CD98 light or
heavy chains.
Because our integrin-CD98 complex was observed in
light membrane fractions and not in dense sucrose
gradient fractions, and because we could not capture the
complex by covalent crosslinking (not shown), the
interaction may not be direct. Nonetheless, an abundance
of evidence points to the functional relevance of this
complex (references above). Furthermore, integrin
αVβ3-CD47 complexes have considerable functional
relevance, despite being found only under relatively mild
detergent conditions (e.g. Brij 58) and exclusively in
the light membrane fractions of a sucrose gradient [ 48
49 ] .
Conclusions
In summary, we show a highly specific biochemical link
between intact β1 integrins and CD98, as demonstrated by
reciprocal co-immunoprecipitation and co-clustering
experiments. Furthermore, CD98 association with β1
integrins appears to occur in the context of an ordered
lipid microdomain (as evidenced by co-localization in light
membrane fractions of sucrose density gradients), and
appears to require the light chain of CD98. These results
may help to explain recent reports that CD98 may modulate
integrin ligand binding, adhesion, signaling, T cell
co-stimulation and cell fusion functions.
Materials and Methods
Antibodies, cell lines, cDNA
Monoclonal antibodies used in this study are: 4F2,
anti-CD98 heavy chain [ 39 ] , A2-2E10, anti-α2 integrin
[ 32 ] ; A3-IIF5, anti-α3 integrin [ 50 ] ; A5-PUJ2,
anti-α5 integrin [ 51 ] ; B5G10, anti-α4 integrin [ 52 ]
; A4-PUJ1, anti-α4 integrin [ 51 ] ; A6-BB, anti-α6
integrin [ 32 ] ; TS2/16, anti-β1 integrin; W6/32,
anti-HLA class I [ 53 ] ; C7, anti-LDL receptor [ 54 ] ;
5E9, anti-CD71 [ 55 ] ; IIIC4, anti-CD 109 [ 32 ] ; 8G6,
anti-CD147/EMMPRIN [ 31 ] ; and KMI6, anti-mouse β1
integrin (Pharmingen, San Diego, CA). The mAb P3 [ 56 ]
was used in all immunoprecipitation and flow cytometry
experiments as a negative control.
Human cell lines used in this study were the
fibrosarcoma cell line HT1080; rhabdomyosarcoma cell line
RD stably transfected with human α3 integrin (RD A3);
lymphoblastoid T cell line Molt4, human mammary
epithelial cell line MTSV 1-7; erythroleukemia K562 cells
and φ nx-Eco packaging cells. K562 cells expressing
integrin α4, α3, or α3 integrin subunit lacking a
cytoplasmic tail (K562-X3C0) were described elsewhere [
30 40 ] . Also utilized were K562 cells expressing α3
D154A, W220A and Y218A mutants [ 41 ] , and Chinese
hamster ovary (CHO) cells that express human α2, α3, α5
or β1 integrin subunits [ 30 32 ] . The cDNA encoding
human CD98 heavy chain [ 57 ] was provided by Jeffrey
Leiden (University of Chicago) in the pUC18 vector. CD98
heavy chain mutants (C109S and C330S) were made by
recombinant PCR technique [ 58 ] with the following
primers: C109Ss - 5'-CGAGCGCCGCGTTCTCGCGAGCTACCGGCG-3';
C109Sas-5'-CGCCGGTAGCTCGCGAGAACGCGGCGCTCG-3'; C330Ss -
5'-GGCAATCGCTGGTCCAGCTGGAGTTTGTCTCAGGC-3';C330Sas -
5'-GCCTGAGACAAACTCCAGCTGGACCAGCGATTGCC-3'. CD98 heavy
chain constructs were subcloned into pLXIZ retroviral
vector (Clontech). Transient transfection of φ nx-Eco
cells and retroviral infection of NIH 3T3 cells was
performed as described [ 59 ] . Stable NIH 3T3 infectants
were selected in growth medium containing 1 mg/ml
zeocin.
Immunoprecipitation experiments
For immunoprecipitations, labeled cellular proteins
were extracted with lysis buffer (1% of the indicated
detergent, 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM PMSF,
10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 mM MgCl2, 0.5
mM CaCl2) for 60 minutes at 4°C. After preincubation with
protein A-sepharose 4CL, the cell extract was incubated
with specific mAb directly conjugated to sepharose 4B for
60 min, washed 3 times with the appropriate lysis buffer
and eluted with Laemmli sample buffer. In some
experiments manganese chloride (2 mM) was added both to
cells (10 minutes prior to lysis) and to the lysis
buffer.
For re-immunoprecipitation experiments, associated
proteins were eluted from immune complexes in 1% Triton
X100 in PBS, then pre-incubated with protein
A-sepharose-4CL. Remaining proteins were then
re-precipitated from the eluate using mAb directly
conjugated to sepharose 4B beads for 30 min at 4°C,
washed three times with 1% Triton X100 in PBS, eluted
with Laemmli sample buffer, and resolved on SDS-PAGE.
Detergent-free purification of light membrane
fractions
Isopycnic fractionation in sucrose gradients was
performed essentially as described [ 43 ] . Briefly,
cells (one confluent T162 flask of HT1080 or NIH 3T3
cells expressing different CD98 constructs) were lysed in
2 ml 500 mM sodium carbonate buffer, pH 11, in the
presence of protease inhibitors and passed through 25
gauge needle 5 times followed by 6 passages through 27
gauge needle. The homogenate (1 ml) was adjusted to 45%
sucrose by addition of 1 ml of 90% sucrose in MBS (25 mM
MES, pH6.5, 150 mM NaCl) and placed at the bottom of an
ultracentrifuge tube. A 5-35% discontinuous sucrose
gradient was formed above (1 ml of 5% sucrose: 2 ml of
35% sucrose, both in MBS containing 250 mM sodium
carbonate) and centrifuged at 45 000 rpm for 16-18 hrs in
SW 55 Ti rotor (Beckman Instruments, Palo Alto, CA). From
the top of each gradient 0.4 ml fractions were collected
and analyzed by SDS-PAGE electrophoresis, or adjusted to
pH 7.5 (with HCl) and used for immunoprecipitation
experiments.
