From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037 and the ** Department of Medicine, University of California San Diego and the Veterans Affairs Healthcare System (VASDHS), San Diego, California 92161
Received for publication, December 13, 2000
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ABSTRACT |
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CD98 is a cell surface heterodimer formed by the
covalent linkage of CD98 heavy chain (CD98hc) with several different
light chains to form amino acid transporters. CD98hc also binds
specifically to the integrin CD98hc is a widely distributed transmembrane protein that was
originally discovered as a T-cell activation antigen (1). CD98hc
expression is tightly linked to cell proliferation, and antibodies
against CD98hc can inhibit cell growth or induce apoptosis in specific
cell types (2, 3). A compelling body of evidence implicates CD98hc in
the transport of amino acids. CD98hc overexpression stimulates multiple
amino acid transport systems including L, y+L, and
xc There is also a growing literature implicating CD98hc in integrin
function. Integrins are heterodimeric adhesion receptors expressed in
almost every multicellular animal cell type (10). Cells can rapidly
modulate their integrins' affinity for extracellular ligands
(activation), thereby regulating multiple
integrin-dependent functions (11). Integrin activation is
inhibited by overexpression of isolated In the present study, we have assessed the relationship between the
amino acid transport and integrin regulatory activities of CD98hc. We
found that the CD98hc alone is necessary and sufficient for the
interaction of CD98 with the Antibodies--
The hybridoma cell line 4F2(C13) (anti-CD98hc)
was purchased from American Type Culture Collection (ATCC, Manassas,
VA). The CD98hc antibody was purified from ascites produced in
pristane-primed BALB/c mice by protein A affinity chromatography. Dr.
S. Shattil (Scripps Research Institute) generously provided the
activation-specific anti- DNA Constructs and Recombinant Proteins--
cDNAs encoding
the expressed integrin cytoplasmic domains joined to four heptad
repeats were cloned into the modified pET-15 vector as described
previously (21). Recombinant expression in BL21 (DE3) pLysS cells
(Novagen) and purification of the recombinant model proteins were
performed in accordance with the manufacturer's instructions
(Novagen), with an additional final purification step on a reverse
phase C18 high performance liquid chromatography column (Vydac).
Polypeptide masses were confirmed by electrospray ionization mass
spectrometry on an API-III quadrupole spectrometer (Sciex; Toronto,
Ontario, Canada), and they varied by less than 4 daltons from those
predicted by the desired sequence.
Tac- Construction of CD98 Chimeras--
HA-NH2 was
constructed by polymerase chain reaction with primers that
included a nine-amino acid influenza hemagglutinin (HA)-tag followed by
a three-Gly linker, which was placed directly after the initiator Met.
The primers used to create HA-COOH added the HA-tag, preceded by a
three-Gly linker to the COOH-terminal portion of CD98hc. All of the
CD98hc chimeras were made by overlap polymerase chain reaction. Cell Culture--
Cell Lysates--
24 h after transfection with CD98hc or one of
the chimeric cDNAs, Affinity Chromatography Experiments--
Recombinant proteins
were expressed in BL21 (DE3) pLysS cells (Novagen) and bound to
His-bind resin (Novagen) through their NH2-terminal His-tag
in a ratio of 1 ml of beads/liter of culture. Coated beads were washed
with PN (20 mM Pipes, 50 mM NaCl, pH 6.8) and
stored at 4 °C in an equal volume of PN containing 0.1% NaN3. Beads were added to cell lysates diluted in buffer A
(0.05% Triton X-100, 3 mM MgCl2, and protease
inhibitors), incubated overnight at 4 °C, and then washed five times
with buffer A. 100 µl of SDS-sample buffer was added to the beads,
and the mixture was heated at 100 °C for 5 min. After centrifugation
at 10,000 rpm for 30 min in a microcentrifuge (5417C Eppendorf),
the supernatant was fractionated by SDS-PAGE and analyzed by
immunoblotting. Chimeras with the extracellular domain of CD69 were
analyzed in the absence of dithiothreitol, because the anti-CD69
antibody employed only detects the nonreduced form of CD69. In some
experiments, proteins were eluted off the beads with 100 µl of
elution buffer (1 M imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9), and 1 ml of immunoprecipitation buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM benzamidine HCl, 1% Triton X-100, 0.05% Tween 20, and
protease inhibitors) was then added. The eluted proteins were
immunoprecipitated overnight at 4 °C with an anti-CD98hc antibody
prebound to protein A-Sepharose beads (Amersham Pharmacia
Biotech). The following day, the beads were washed three times
with the immunoprecipitation buffer and heated in sample buffer for
SDS-PAGE containing 1 mM dithiothreitol. Samples were
separated on 4-20% SDS-polyacrylamide gels (Novex) and transferred to
nitrocellulose membranes. Membranes were blocked with 5% nonfat milk
powder in Tris-buffered saline and stained with streptavidin peroxidase
or with specific antibodies and appropriate peroxidase conjugates.
Bound peroxidase was detected with an enhanced chemiluminescence kit
(Amersham Pharmacia Biotech). Equal loading of Ni2+ beads
with recombinant proteins were verified by Coomassie Blue staining of
SDS-PAGE profiles of SDS-eluted proteins.
Flow Cytometry--
Analytical two-color flow cytometry was
performed as described previously (24). PAC1 binding was assessed in a
subset of transiently transfected Amino Acid Transport--
3H-Labeled Ile (77 Ci/mM)
was purchased from Amersham Pharmacia Biotech. CD98 Heavy Light Chain Association Is Not Required for the
Interaction of CD98 with Integrins--
CD98 is a heterodimer formed
by a common heavy chain (CD98hc) disulfide-bonded to one of a number of
light chains that mediate amino acid transport. CD98hc has two
extracellular cysteines, one of which (Cys109) is involved
in covalent association of the heavy and light chains (25). To examine
the role of CD98 heavy-light chain association on its interactions with
integrins, we first investigated the effect of mutation of these
cysteines (Cys109 and Cys330). The C109S
(C1) mutation alone, or in combination with the C330S (Cless) mutation,
is reported to reduce the amino acid transport activity of CD98 (7, 25,
26). However, the capacity of CD98hc to complement dominant suppression
was not impaired by mutation of both or either cysteines (Fig.
1A). We confirmed that the
mutant lacking cysteines (Cless) failed to form stable disulfide-bonded heterodimers with CD98 light chains (Fig. 1C) and found that
it, like wild-type CD98hc, bound to the integrin
As an alternative approach to evaluate the role of the CD98 heavy-light
chain association, we examined the effect of increased expression of a
CD98 light chain (hLAT1) on integrin interactions. When CHO cells were
transfected with CD98hc, there was a substantial quantity of free heavy
chain (Fig. 2B). Transfection
of increasing quantities of a light chain, hLAT1, resulted in an
increasing proportion of CD98 heterodimers (Fig. 2B). As
expected, formation of increased CD98 heterodimers led to a marked
increase in amino acid transport (Fig. 2A). However,
increased heterodimer formation did not detectably alter the ability of
CD98hc to bind to The NH2 Terminus of CD98hc Is Intracellular, and the
COOH Terminus Is Extracellular--
In the foregoing experiments, we
found that the interactions of CD98 with integrins could be ascribed to
its heavy chain. Consequently, we wished to analyze the structural
determinants in the heavy chain responsible for these interactions.
CD98hc is predicted to be a type II transmembrane protein, with the
COOH terminus extracellular and the NH2 terminus
intracellular. To document the membrane topography of CD98hc, the
NH2 and COOH terminus of CD98hc were separately tagged
using a HA-tag. When cells were transfected with the
COOH-terminal-tagged cDNA, the epitope tag was readily detected on
the cell surface as indicated by a rightward shift in the fluorescence
intensity (Fig. 3A). In
contrast, the amino-terminal tag was not detected on the cell surface.
The presence of the epitope tag on the NH2 or COOH terminus
did not effect the overall surface expression of CD98hc as measured
with an anti-CD98hc antibody (Fig. 3A). Furthermore, both
the NH2- and COOH-terminal tags were present on the
expressed protein (Fig. 3B). Consequently, we conclude that
the NH2 terminus of CD98hc is intracellular, and the COOH
terminus is extracellular.
The Cytoplasmic and Transmembrane Domains of CD98hc Are Required
for Its Interaction with Integrins--
Having confirmed the membrane
topography of CD98hc, we wished to examine the role of its cytoplasmic
and extracellular domains in the integrin interaction. We first deleted
the cytoplasmic domain of CD98hc (Fig.
4A). This abolished its
ability to complement dominant suppression (Fig. 4B),
although it did not block surface expression (data not shown, but see
Fig. 5). Deletion of the CD98hc cytoplasmic domain completely abolished its ability to bind to the
To assess whether the CD98hc cytoplasmic domain was sufficient for this
interaction, additional chimeric exchanges were made. A construct
containing the extracellular and transmembrane domains of CD69
(C98T69E69) joined to the
cytoplasmic domain of CD98hc failed to bind to the Amino Acid Transport Activity and CODS Require Structurally
Distinct Regions of CD98hc--
The foregoing studies established that
both the cytoplasmic and transmembrane domains of CD98hc were required
for its capacity to bind to the CD98hc combines with several different light chains to form a
series of heterodimers that are involved in amino acid transport. CD98hc binds to integrin The formation of a covalent CD98 heterodimer is not required for its
effects on integrin function. CD98hc has two extracellular cysteines
Cys109 and Cys330. Cys109 is near
the transmembrane domain of CD98hc and results in a disulfide bridge
with a cysteine in an extracellular loop of the light chain between
transmembrane domains 3 and 4 (25). Mutation of Cys109 and
Cys330 disrupted the covalent association with the light
chain but did not impair interactions with or effects on integrins.
While the covalent association was lost, it is possible that there was
still a noncovalent interaction. Indeed, Pfeiffer et al.
(25) reported that the C109S mutant does still support the surface
expression of the light chain. The C109S mutation still displays the
same transport characteristics as the disulfide-bound heterodimers, albeit at a reduced rate. Moreover, we also found that overexpressed free heavy chains could also bind to the The cytoplasmic and transmembrane domains of CD98hc are both necessary
and sufficient for binding to the integrin Regulation of amino acid transport and integrins by CD98hc is a
distinct and separable function of the polypeptide. Chimeras in which
the cytoplasmic or transmembrane domains of CD98hc were replaced with
those of CD69 lost the capacity to bind to CD98hc functions as a chaperone to bring the associated light chains
(LAT1, LAT2, y+LAT1, y+LAT2, xCT, and asc-1) to
the plasma membrane (29, 30). We found that the interaction of CD98hc
with integrins and amino acid transporters are ascribable to distinct
domains of the protein and are not mutually exclusive.
Integrin-mediated adhesion often leads to the polarization of these
receptors to the adherent cell surface. Consequently, the
integrin-CD98hc interaction may serve to polarize the localization of
CD98 and, consequently, amino acid transport. Conversely, CD98hc can
influence multiple integrin-dependent functions, including
virus-induced cell fusion, T-cell costimulation, and cell adhesion (13,
15-17). Thus, the CD98hc-integrin association can promote
integrin-mediated cell adhesion that, in turn, could serve to localize
the activities of CD98hc-linked amino acid transporters.
1A cytoplasmic domain
and regulates integrin function. In this study, we examined the
relationship between the ability of CD98hc to stimulate amino acid
transport and to affect integrin function. By constructing chimeras
with CD98hc and a type II transmembrane protein (CD69), we found that
the cytoplasmic and transmembrane domains of CD98hc are required for its effects on integrin function, while the extracellular domain is
required for stimulation of isoleucine transport. Consequently, the
capacity to promote amino acid transport is not required for CD98hc's
effect on integrin function. Furthermore, a mutant of CD98hc that lacks
its integrin binding site can still promote increased isoleucine
transport. Thus, these two functions of CD98hc are separable and
require distinct domains of the protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(4). Furthermore, mutations in its closest
paralogue, D2 (r-BAT), lead to a disorder of cysteine transport (5).
Structurally, CD98 is a disulfide-bonded heterodimer of a common
~80-kDa heavy chain (CD98hc) with one of several ~40-kDa light
chains. Because these light chains have multiple membrane-spanning
domains, they resemble permeases and are believed to provide the
amino acid transport activity of CD98 (6-8). CD98hc may act to
regulate the expression and cellular localization of the amino acid
transporting activity of the light chain (6, 9). Thus, this widely
distributed membrane protein is strongly implicated in amino acid transport.
1A integrin
cytoplasmic domains (dominant suppression) (12). CD98hc was identified
as an integrin regulator in an expression-cloning scheme for proteins
that can complement dominant suppression
(CODS)1 (13). In addition,
CD98hc binds to integrin
1A cytoplasmic domains, and
this interaction correlates with CODS (14). Furthermore, clustering
CD98hc activates multiple integrin-dependent functions (13,
15, 16) and mimics
1 integrin cosignaling in T-cells (17). Thus, CD98hc physically and functionally interacts with integrin
adhesion receptors. Indeed, clustering of CD98hc can stimulate several
classes of integrins in multiple cell types (13, 15, 16).
1A integrin tail and for CODS. By forming chimeras between CD98hc and another type II
transmembrane protein, we found that the cytoplasmic and transmembrane
domains of CD98hc are required for integrin interactions but not for
stimulation of isoleucine transport. In contrast, the CD98hc
extracellular domain was required for stimulation of amino acid
transport. Thus, the amino acid transport activity and integrin
interactions of CD98 are independent activities of the protein and are
mediated by different domains of CD98hc.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIb
3 monoclonal
antibody, PAC1 (18). The
anti-
IIb
3-activating monoclonal antibody,
anti-LIBS6, has been described previously (19). The anti-Tac antibody,
7G7B6, was obtained from the ATCC and was biotinylated with
biotin-N-hydroxysuccinimide (Sigma) according to
manufacturer's instructions. The
IIb
3-specific peptide inhibitor,
Ro43-5054(20) was a generous gift from B. Steiner (Hoffmann-La Roche,
Basel, Switzerland). The mouse hybridoma cell line, 12CA5 (anti-HA),
was purchased from ATCC. The anti-CD69 antibody (C1, a rabbit
polyclonal antibody) was a generous gift from Drs. F. Diaz-Gonzalez and
F. Sanchez-Madrid (University of Madrid, Spain).
5 and Tac-
1A DNA in modified CMV-IL2R
expression vectors (22) were generously provided by Drs. S. LaFlamme
and K. Yamada (National Institutes of Health). Human 4F2 antigen
(CD98hc) cDNA was provided by Dr. J. M. Leiden (University of
Chicago, Chicago, IL) and was subcloned into pcDNA1 as an
EcoRI fragment. Cless (C109S,C330S), C1 (C109S), and
C2 (C330S) mutants of CD98hc were gifts from Dr. M. Palacin
(Universitat de Barcelona). cDNA encoding hLAT1, a CD98 light
chain, was a gift from Dr. F. Verrey (University of Zurich).
CD98
deletes amino acids 2-77 (all numbering uses the amino acid sequence
reported in entry 4F2_human (entry P08195) of the Swiss-Prot data base
as of November 2000), which removes the entire cytoplasmic domain of
CD98hc, maintaining the initiator methionine as well as the presumptive
stop transfer sequence Val-Arg-Thr-Arg.
C69T98E98 contains amino acids
1-38 of CD69 (Swiss-Prot accession number Q07108) and amino acids 82-529 of CD98hc (Swiss-Prot accession number P08195).
C98T69E98 contains amino acids
amino acids 1-81 and 105-529 of CD98hc and amino acids 39-64 of
CD69. C98T98E69 contains amino
acids 1-104 of CD98hc and amino acids 65-199 of CD69.
C98T69E69 contains amino acids
1-81 of CD98hc and amino acids 39-199 of CD69.
Py cells, a CHO cell line
expressing a constitutively active recombinant chimeric integrin
containing the extracellular and transmembrane domains of human
IIb
3 fused to the cytoplasmic domains of
6A
1
(
IIb
6A
3
1)
(23), were maintained in Dulbecco's modified Eagle's medium
(BioWhitaker) supplemented with 10% fetal calf serum (BioWhitaker),
1% nonessential amino acids (Life Technologies, Inc.), 1% glutamine
(Sigma), 1% penicillin and streptomycin (Sigma), and 700 µg/ml G418
(Life Technologies, Inc.).
Py cells were washed twice in
phosphate-buffered saline and surface-biotinylated using
Sulfo-Biotin N-hydroxysuccinimide in
phosphate-buffered saline according to the manufacturer's instructions (Pierce). They were then washed twice with Tris-buffered saline and
lysed by sonication on ice in buffer A (1 mM
Na3VO4, 50 mM NaF, 40 mM sodium pyrophosphate, 10 mM Pipes, 50 mM NaCl, 150 mM sucrose, pH 6.8) containing 1%
Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA, and
protease inhibitors (aprotinin, 5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and clarified by centrifugation.
Py cells (cells positive
for cotransfected Tac-
5 or Tac-
1 as
measured by 7G7B6 binding). Integrin activation was quantified as an
activation index (AI) defined as (F
Fo)/(FLIBS6
Fo) in which
F is the median fluorescence intensity of PAC1 binding,
Fo is the median fluorescence intensity of PAC1
binding in the presence of saturating concentration of a competitive
inhibitor (1 µM Ro43-5054), and FLIBS6 is the maximal median fluorescence intensity in the presence of the integrin activating antibody, anti-LIBS6 (2 µM). Percent reversal
was calculated as (AI(
1A + CD98)
AI
1A)/(AI
5
AI
1A). AI
1A is the activation index of cells transfected with Tac-
1A
chimeras, AI(
1A + CD98) is the AI of cells
cotransfected with CD98hc and Tac-
1A chimeras, and
AI
5 is the AI of cells transfected with
Tac-
5.
Py cells
were transfected with cDNAs encoding CD98hc or one of the chimeric
cDNAs in the presence or absence of cDNA encoding hLAT1 using
LipofectAMINE 2000 (Life Sciences Technologies). Transport studies were
performed on cells that were transfected with 80-90% efficiency, as
judged by staining for CD98 in flow cytometry. Before the transport
assays, cells were rinsed with warm Na+-free Hanks'
buffered salt solution (HBSS) (136.6 mM ChCl, 5.4 mM KCl, 4.2 mM NaHCO3, 2.7 mM Na2HPO4, 1 mM
CaCl2, 0.5 mM MgCl2, 0.44 mM KH2PO4, 0.41 mM
MgSO4, pH 7.8), in which the sodium-containing salts were
iso-osmotically replaced with choline, to remove extracellular Na+ and amino acids. Cells were equilibrated in warm
choline-substituted HBSS for 10 min. The uptake of radiolabeled amino
acids (2 µCi of [3H]Ile/ml) at 100 µmol/liter in 1 ml
of choline-substituted HBSS was measured for 20 s at 37 °C.
Uptake of [3H]Ile was linearly dependent on incubation
time up to at least 3 min. Uptake was terminated by washing the cells
rapidly four times with 1 ml/well of ice-cold HBSS. The cells were
lysed overnight with 1 ml 2 M NaOH. A 0.95-ml aliquot from
each well was mixed with scintillation fluid, and radioactivity was
quantified in a Beckman LS 6000SC liquid scintillation counter. The
remaining 0.1 ml was analyzed for protein content using the BCA protein assay reagent (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1A
cytoplasmic domain (Fig. 1B). Thus, covalent CD98
heterodimer formation is dispensable for the interaction of CD98hc with
the integrin
1A cytoplasmic domain and for CODS.
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Fig. 1.
Stable formation of the CD98 heterodimer is
not required for its effects on integrin function. A,
effect of cysteine mutations on CODS. Py cells were
transfected with Tac-
1 and either wild-type CD98hc or
CD98hc with serine substitutions at cysteines (Cys109,
Cys330) involved in covalent heterodimer formation.
Twenty-four h after transfection, cells were collected and the
Tac-positive subset of cells was analyzed for the ability to bind to
the PAC1 antibody. Data are expressed as percentage reversal of
Tac-
1 suppression, which is calculated as 100 × (AITac-
1 + CD98
AITac-
1)/(AITac-
5
AITac-
1). AI is the activation index of
cells transfected with Tac-
1 alone or in combination
with CD98hc or with Tac-
5. Cless = CD98hc
(C109S,C330S), C1 = CD98hc (C109S), C2 = CD98hc
(C130S). B, CD98hc Cys109 and Cys130
are not required for binding to the integrin
1A
cytoplasmic domain.
Py cells were transfected with either
CD98hc, the Cless mutant, or vector DNA. After 24 h surface
proteins were labeled with Sulfo-Biotin
N-hydroxysuccinimide, cell lysates were incubated with beads
coated with model proteins containing
1A or
IIb cytoplasmic tails. The bound proteins were eluted
and immunoprecipitated with anti-CD98hc and fractionated by reduced
SDS-PAGE. Biotinylated CD98hc was identified by
streptavidin-peroxidase-generated chemiluminescence staining of an
~80-kDa polypeptide. In addition, the starting cell lysate was
immunoprecipitated with either anti-CD98hc antibodies
(Lysate) or with a control IgG (IgG). Proteins
were fractionated by reduced SDS-PAGE, and biotinylated proteins were
detected by streptavidin-peroxidase-generated chemiluminescence.
C, mutation of CD98hc cysteines 109 and 130 disrupts
covalent heterodimer formation. Biotinylated lysates of surface labeled
cells transfected with either CD98hc or Cless CD98hc were
immunoprecipitated with anti-CD98hc antibodies as described in
B. The lysates were fractionated by SDS-PAGE in the absence
or presence (+DTT) of reducing agent. Biotinylated
polypeptides were identified by streptavidin-peroxidase-generated
chemiluminescence.
1A cytoplasmic tails (Fig.
2C). Thus, while the formation of CD98 heterodimers is
important for the stimulation of amino acid transport, CD98hc alone is
sufficient for binding to the integrin
1A tail and for
CODS.
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Fig. 2.
Effect of coexpression of the hLAT1 light
chain on amino acid transport, heterodimer formation, and the binding
of CD98hc to the 1A cytoplasmic
domain. A, amino acid transport:
Py cells
were transfected with cDNAs encoding CD98hc, hLAT1, CD98hc plus
hLAT1 or vector DNA and assayed for the uptake of
[3H]isoleucine after 24 h. Ile uptake was measured
in a Na+-free solution, and the values are expressed as
cpm/mg of protein. B, heterodimer formation: CHO cells were
transfected with cDNA encoding CD98hc (4 µg) plus increasing
amounts of hLAT1 light chain (0, 2, 6 µg). Cell surface proteins were
biotinylated, and CD98 was immunoprecipitated from cell lysates with an
anti-CD98hc antibody. Heterodimers were visualized by running
nonreduced SDS-PAGE as described in the legend to Fig. 1C.
C, binding to the
1A cytoplasmic domain: CHO cells were
transfected with cDNA encoding CD98hc and increasing amounts of
hLAT1. Twenty-four h later cells were surface-biotinylated and lysed.
Lysates were mixed with beads coated with
1A or
IIb tails. Beads were washed, bound, and eluted proteins
were immunoprecipitated with anti-CD98hc antibody and fractionated by
reduced SDS-PAGE. The biotinylated polypeptides were detected by
streptavidin-peroxidase chemiluminescence.
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Fig. 3.
The NH2-terminal domain of CD98hc
is cytoplasmic. CHO cells were transfected with CD98hc HA-tagged
either at the NH2 terminus (HA-NH2)
or at the COOH terminus (HA-COOH). A, twenty-four
h after transfection cells were with stained with either an anti-HA
antibody (top panels) or with anti-CD98hc antibody, 4F2
(bottom panels), and analyzed by flow cytometry.
B, expression of HA tags: CHO cells transfected as in
A were lysed and immunoprecipitated with an anti-CD98hc
antibody (IP) or normal mouse IgG (IgG). The
immunoprecipitates were fractionated by reduced SDS-PAGE. The
immunoblots were stained with an anti-HA antibody and developed by
peroxidase-mediated chemiluminescence.
1A cytoplasmic domain (Fig. 4C). As an
alternative approach, we exchanged the cytoplasmic domain of CD98hc
with another type II transmembrane protein (CD69). That chimera
(C69T98E98) failed to complement
dominant suppression (Fig. 4B) and failed to bind to the
1A cytoplasmic domain (Fig. 4C). Thus, the
cytoplasmic domain of CD98hc is required for its capacity to interact
with integrins.
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Fig. 4.
The cytoplasmic domain of CD98hc is required
for integrin interactions. A, model of CD98hc/CD69
chimeras. B, complementation of dominant suppression (CODS):
Py cells were transfected with Tac-
1 and
the CD98hc constructs depicted in A. Twenty-four h after
transfection, cells were collected and the Tac-positive subset of cells
were analyzed for the ability to bind to the PAC1 antibody. Data are
expressed as percentage reversal of Tac-
1A suppression,
as described in the legend to Fig. 1. C, binding to
1A tail: affinity chromatography with
1A
or
IIb tails was performed using lysates of
surface-biotinylated CHO cells that had been transfected with the
constructs described in A. Bound proteins that contain the
extracellular domain of CD98hc (panels a, b, and
c) were eluted from the beads, immunoprecipitated with
anti-CD98hc antibody, fractionated by SDS-PAGE, and the biotinylated
polypeptides were detected by streptavidin-peroxidase
chemiluminescence. Bound proteins that contain the extracellular domain
of CD69 (panel d) were fractionated by SDS-PAGE, and CD69
was detected by immunoblot with anti-CD69 antibodies.
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Fig. 5.
The cytoplasmic and transmembrane domains of
CD98hc are necessary and sufficient for binding to
1A cytoplasmic domains. CHO cells
were transfected with each of the chimeric cDNAs depicted on the
left-hand side of the figure. Twenty-four h later, surface
proteins were labeled with Sulfo-Biotin
N-hydroxysuccinimide, and the cells were lysed. Cell lysates
were incubated with beads coated with model proteins containing
1A or
IIb cytoplasmic tails. Bound and
eluted proteins that contain the extracellular domain of CD98hc
(CD98hc, C98T69E98) were
immunoprecipitated with anti-CD98hc antibody and fractionated by
reduced SDS-PAGE, and the biotinylated polypeptides were detected by
streptavidin-peroxidase chemiluminescence. Bound proteins that contain
the extracellular domain of CD69 (CD69,
C98T69E69,
C98T98E69) were detected by
immunoblot with the anti-CD69 antibody.
1A
cytoplasmic domain (Fig. 5). However, addition of the transmembrane
domain of CD98hc (C98T98E69)
resulted in binding that was comparable with that observed with
full-length CD98hc. To test whether the transmembrane domain of CD98hc
was required for binding to integrin tails, a construct was made in which only the transmembrane domain was replaced with that of CD69 and
the extracellular and cytoplasmic domains were retained (C98T69E98). That construct also
failed to bind to the
1A cytoplasmic domain (Fig. 5).
Thus, binding of CD98hc to the
1A cytoplasmic domain
requires both its cytoplasmic and transmembrane domains.
1A cytoplasmic domain.
To investigate whether the functional effects of CD98hc correlated with
its binding to the
1A cytoplasmic domain, each of these
chimeras (Fig. 6A) was analyzed for its capacity to mediate CODS and to promote Ile transport. In these experiments, the expression of each chimera was verified by
flow cytometry, and equivalent expression was observed for each one
(data not shown). As previously noted, the substitution of the
cytoplasmic domain of CD98hc with that of CD69 abolished CODS
(C69T98E98; Fig. 6B).
However, this chimera stimulated Ile transport to comparable
levels with wild-type CD98hc (Fig. 6C). Similarly,
substitution of the transmembrane domain of CD98hc (C98T69E98) markedly suppressed
effects on integrin function (Fig. 6B) but had little effect
on the ability to stimulate Ile transport (Fig. 6C). Thus,
the amino acid transport activity of CD98hc is not sufficient for CODS.
Conversely, the substitution of the extracellular domain of CD98hc with
that of CD69 (C98T98E69) preserved
effects on integrin function (Fig. 6B) but abolished amino
acid transport activity (Fig. 6C). Thus, the extracellular
domain of CD98hc is necessary (in the context of another type II
transmembrane protein) for its ability to stimulate isoleucine
transport. Conversely, the transmembrane and cytoplasmic domains of
CD98hc are necessary and sufficient for binding to the
1A tail and for augmentation of integrin function.
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Fig. 6.
The cytoplasmic and transmembrane domains of
CD98hc are necessary and sufficient for its effect on integrin
function, but not amino acid transport. A, model of
CD98hc/CD69 chimeras. B, CODS: Py cells were
transfected with Tac-
1 and the CD98hc chimeras depicted
in A. Twenty-four h after transfection, cells were
collected, and the Tac-positive subset of cells were analyzed for the
ability to bind to the PAC1 antibody. Data are expressed as percentage
reversal of Tac-
1 suppression, as described in the
legend to Fig. 1. C, amino acid transport:
Py
cells were transfected with cDNAs encoding the hLAT1 light chain
and the CD98hc chimeras depicted in A. The uptake of
[3H]isoleucine was measured 24 h after transfection
as described under "Experimental Procedures." The uptake was
measured in a Na+-free solution, and the values are
expressed as cpm/mg protein, where the base-line uptake in cells
transfected with hLAT1 alone has been subtracted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1A cytoplasmic domains and
blocks the capacity of
1A cytoplasmic domains to
suppress integrin activation (CODS). We have compared the structural
requirements of CD98hc for interaction with integrins with those
involved in regulation of amino acid transport. Here we report that: 1)
mutation of cysteines that disrupt CD98 heavy-light chain association
and reduce amino acid transport do not disrupt its binding to
1A or its effect on integrin activation. 2) The
cytoplasmic and transmembrane domains of CD98hc fused to another type
II transmembrane protein are both necessary and sufficient for binding
to the integrin
1A tail and for CODS. This chimera failed to
stimulate amino acid transport. 3) Replacement of the cytoplasmic or
transmembrane domains of CD98hc with those of CD69 blocked the capacity
of CD98hc to bind to
1A and regulate integrin
activation, but had minimal effects on the amino acid transport
function of CD98. Thus, the amino acid transport function of CD98 is
not required for its effects on integrin function, and amino acid
transport can occur in the absence of CD98hc-integrin association.
1A tail.
Furthermore, cotransfection of the hLAT1 light chain increased
formation of heterodimers and amino acid transport but did not augment
integrin interactions or effects. Consequently, our results indicate
that the covalent association of CD98hc with a light chain is not
required for its interaction with integrins or for the functional
regulation of integrins.
1A tail and
for effects on integrin function. When either of these domains was
removed from CD98hc, integrin interactions were lost. Conversely, effects on integrins could be conveyed to CD69 by addition of these two
domains. What is the role of the CD98hc transmembrane domain in binding
to the
1A cytoplasmic tail? It is possible that the
CD98hc transmembrane domain influences the conformation of the
cytoplasmic domain to promote binding to integrin cytoplasmic domains.
Alternatively, our integrin cytoplasmic domain model protein was based
on that predicted from the sequence (ITB1_human) in the Swiss-Prot data
base (Entry P05556). Glycosylation mapping studies have suggested that
1A (Lys752-Ile757) of the
predicted "cytoplasmic" domain may reside in the membrane (27).
Consequently, the CD98hc transmembrane domain may directly interact
with a transmembrane portion of our model protein "tail." Furthermore, other integrin-binding proteins, such as cytohesin-1, Rack1, and skelemin also bind the membrane proximal region (28). Thus,
the localization of this region in the membrane may specify preferential binding of integrin-associated proteins. Finally, the
CD98hc solubilized from membranes could be associated with other
proteins via the transmembrane domains. These "adapters" might
contribute to the CD98hc-
1A tail interaction. In any
case, our studies provide the first delineation of a specific
functional role for the cytoplasmic and transmembrane domains of
CD98hc, interaction with and regulation of
1A integrin function.
1A and regulate integrin activation. In contrast, these replacements had
little effect on the amino acid transport function of CD98. Conversely,
the exchange of the extracellular domain of CD98hc with that of CD69
resulted in a protein that was still capable of affecting integrin
function but did not stimulate isoleucine transport. Thus, the amino
acid transport activity of CD98hc is not required for its effect on
integrin function.
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ACKNOWLEDGEMENTS |
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We thank our colleagues for their generosity in providing the reagents listed under "Experimental Procedures." We thank Drs. François Verrey and Manuel Palacin for reagents and for helpful discussions.
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FOOTNOTES |
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* This work was supported by Grants HL48728, HL59007, and AR27214 from the National Institutes of Health. This is publication number 13766-VB.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.
These authors contributed equally to this work.
§ Supported by The United States Army Medical Research and Materiel Command under DMAD 17-97-1-7056.
¶ Young Investigator Award of the National Kidney Foundation. Current address: Dept. of Medicine, Vanderbilt University, S-3223 Medical Center North, Nashville, TN 37232-2372.
Supported by grants from the Wellcome Trust and Susan G. Komen
Breast Cancer Foundation.
Supported by National Institutes of Health Grant DK 28602.
§§ Supported by grants from the Veterans Affairs Medical Research Service.
¶¶ To whom correspondence should be addressed. Tel.: 858-784-7143; Fax: 858-784-7343; E-mail: ginsberg@scripps.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M011239200
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ABBREVIATIONS |
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The abbreviations used are: CODS, complement dominant suppression; CHO, Chinese hamster ovary; Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; AI, activation index; HBSS, Hanks' buffered salt solution.
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