(Received for publication, March 20, 1995; and in revised form, April 26, 1995)
From the
We screened monoclonal antibodies to cell-surface proteins and
selected an antibody, called 6H1, that recognizes a putative
integrin-associated protein. The 6H1 monoclonal antibody (mAb)
indirectly coprecipitated The adhesion of cells to the extracellular matrix is dependent
on the function of integrins, a large family of ubiquitous
heterodimeric cell-surface proteins(1) . Cell attachment to
extracellular matrix through integrins results in generation of
biochemical signals (e.g. changes in protein phosphorylation,
intracellular pH, and Ca Notably, different integrins binding to the same ligands may induce
distinct post-ligand binding
events(5, 6, 7) , consistent with specific
signaling pathways. Although the mechanisms for specific
integrin-mediated signal transduction remain largely unexplored,
different integrin Only a few
integrin-associated proteins have been identified that could
potentially facilitate specific signaling. A 50-kDa protein
(IAP-50/CD47), associated with a This study is aimed at
identifying proteins that associate selectively with particular
For
reprecipitation of CD63 protein, HT1080 cells (7
Of more than 600 hybridoma
supernatants screened by immunoprecipitation, 131 precipitated various
cell-surface proteins but failed to precipitate or coprecipitate any
molecules resembling integrins. Another 28 antibodies precipitated
biotin-labeled material that appeared on SDS-PAGE as two protein bands
of sizes characteristic for integrin From a
biotinylated extract of HT1080 cells, the 6H1 antibody
immunoprecipitated two integrin-like proteins of 150 and 130 kDa (Fig. 1, lanea). In addition, this antibody
coprecipitated a 67-kDa protein, similar in size to one of the bands
coprecipitated with
Figure 1:
Coimmunoprecipitation of an
integrin-like heterodimer by mAb 6H1. HT1080 cells were surface labeled
with biotin, lysed in 1% Brij 96 buffer, and immunoprecipitated with
mAb 6H1 (lanea), mAb to the indicated integrins (lanesb-e), or with the P3 negative control (lanef). Precipitates were resolved in 9% SDS-PAGE
under non-reduced conditions.
When analyzed by flow cytometry with a saturating
amount of mAb (100 µg/ml), the 6H1 antigen was detected on the
surface of K562, K562-A2, K562-A3, and K562-A6 cells at approximately
similar levels, indicating that expression was not specifically
dependent on the presence of the
Figure 2:
The 6H1 mAb fails to recognize human
integrin subunits on hamster or mouse cells. CHO-A2, CHO-A3, CHO/B2-A5,
CHO-B1, and P388-A6 cells were stained with control mAb P3 (firstcolumn), mAb 6H1 (secondcolumn), or
anti-integrin mAbs (thirdcolumn), and expression was
analyzed by flow cytometry. Anti-integrin mAbs were:
anti-
The
6H1 antigen was expressed on the surface of all human cell types
analyzed as well as platelets (not shown). Notably, the 6H1 antibody
stained a variant of JY cells that lacks all To determine whether any proteins immunoprecipitated by
the 6H1 mAb indeed represent integrin
Figure 3:
Reprecipitation of
The
apparent specific association of the 6H1 antigen with the
Figure 4:
Selective coprecipitation by the 6H1 mAb
of integrins from K562 cell transfectants. Equal numbers of K562,
K562-A2, K562-A3, and K562-A6 cells were surface labeled with biotin,
lysed in 1% Brij 96 buffer, and immunoprecipitated with either 6H1 mAbs (lanesd-f, h) or with anti-a
Figure 5:
Biochemical characterization of the 6H1
antigen. HT1080 cells were surface labeled with biotin (lanesa and b) or metabolically labeled with
[
To further characterize
the 45-60 kDa antigen, 5-10 pmol of this protein was
purified from K562 cells using a 6H1 immunoaffinity column, and the
sequence of the first 30 amino-terminal amino acids was determined (Fig. 6). A search of protein sequences available from the
GenBank data base revealed a perfect match between the 6H1 antigen and
residues 2-31 from the amino terminus of the CD63 protein, except
for three missing cysteines. Consistent with this result, both 6H1 and
a previously characterized anti-CD63 mAb called RUU-SP 2.28
precipitated a very similar pattern of major and minor
Figure 6:
Amino-terminal sequence of the 6H1
antigen. The 6H1 antigen was immunopurified from K562 cells, the
amino-terminal sequence was determined as described under
``Materials and Methods,'' and this sequence was aligned with
the sequence from the CD63 antigen obtained from GenBank. No
determination could be made at three positions (indicated by
``-''), corresponding to cysteine residues in
CD63.
Figure 7:
The
6H1 mAb and a known anti-CD63 mAb yield similar patterns of
coprecipitating proteins. HT1080 cells were surface labeled with
biotin, lysed in 1% Brij 96 buffer, and immunoprecipitated with
anti-
To further
demonstrate specific CD63 association with
Figure 8:
Detection of CD63 in an
Figure 9:
Colocalization of CD63 and
Figure 10:
Role of the
Specific interactions of particular Our detection
of CD63-integrin associations is unlikely to result simply from
spurious cross-reactivity of anti-CD63 antibodies with integrins.
First, the 6H1 mAb to CD63 failed to coprecipitate
The specificity
and relevance of the CD63-integrin interactions are apparent from
several different results. First, in multiple cell lines, CD63
associated with The 6H1 antigen is identical to the CD63 protein, based on
amino-terminal sequence identity. Consistent with this, 6H1 and an
established anti-CD63 mAb (RUU-SP 2.28) both 1) specifically
coprecipitated The ability of CD63 to complex
specifically with The CD63 protein has been separately
described as a platelet activation antigen and as a stage-specific
melanoma membrane antigen, ME-491(41, 49) . It belongs
to the TM4 (or tetraspans) family of proteins, which includes CD9,
CD37, CD53, CD81 (TAPA-1), CD82 (C33/IA4 antigen), and some other
proteins(17) . These all contain four putative transmembrane
domains and show some similarity in their primary
structures(50) . As we have confirmed, CD63 appears on the
surface of most cultured cell lines at a moderate level(18) .
In addition, it is incorporated into the membranes of different types
of intracellular granules, including
lysosomes(18, 39, 41) , endothelial
Weibel-Palade bodies(51) , platelet-dense
granules(52) , and the major histocompatibility complex class
II compartment(53) . The CD63 molecule can be rapidly delivered
to the cell surface after cells are treated (activated) with various
stimuli(41, 54) . Immunofluorescence staining of
Because we have not yet found a cell line lacking CD63, we have been
unable to carry out definitive transfection experiments to address the
functional implications of CD63- In
several experiments, a similar pattern of additional proteins was
coprecipitated by antibodies to both CD63 and In conclusion, we have identified novel
protein-protein associations that are highly specific for a subset of
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and/or
, but not
, or
from Brij 96 detergent lysates of multiple cell lines. Large
scale purification using the 6H1 mAb yielded a single protein of
45-60 kDa with an amino-terminal sequence that exactly matched
CD63. Confirming that the 6H1 mAb recognized the CD63 protein, 6H1 and
a known anti-CD63 mAb yielded identical coprecipitation results and
identical colocalization into lysosomal granules containing cathepsin
D. Furthermore, we used an established anti-CD63 mAb to detect this
protein in an
immunoprecipitate, and
also we observed VLA-3 and CD63 colocalization in cellular
``footprints.'' Notably, the cytoplasmic domain of
was neither required nor sufficient for CD63
association, suggesting that it occurred elsewhere within the
complex. Knowledge of these specific
CD63-
and
CD63-
biochemical associations should
lead to critical insights into the specialized functions of
,
,
and CD63.
levels) and can regulate a
variety of biological responses including cell migration,
proliferation, apoptosis, and
differentiation(1, 2, 3, 4) .
and
chain cytoplasmic domains have been
associated with particular post-ligand binding
events(5, 8, 9, 10, 11) .
Because integrins themselves lack obvious enzymatic activity, we
hypothesize that their differential associations with other proteins
might be important for specific integrin signaling.
-like integrin, is
essential for neutrophil phagocytosis enhanced by fibronectin but not
by laminin(12) . The phosphorylation of other
-associated proteins occurs in response to
platelet-derived growth factor (13) or insulin (14) stimulation. However, no proteins have yet been identified
that specifically associate with particular
integrins. Cytoskeletal proteins such as
-actinin and talin
seem to associate with many different integrins, including those
containing
,
, and
subunits (15, 16) .
integrins. We used mild immunoprecipitation
conditions to select for a new mAb
(
)against a
protein specifically associated with the
and
integrins. This protein
was subsequently identified as the CD63 antigen, a member of the TM4
(or tetraspans) family of proteins(17) . Relatively little is
known regarding CD63 other than that it is widely expressed on the
surface of many cell types (18) and is present in the membranes
of many types of intracellular granules (see ``Discussion'').
Cell Lines
The human fibrosarcoma cell
line HT1080, the human erythroleukemia cell line K562, and the various
K562 transfectants were cultured in RPMI 1640 media supplemented with
10% fetal bovine serum (FBS). The K562 transfectants K562-A2 and
K562-A3 were transfected with (19) or
(20) cDNA as previously described. To make
K562-A6 cells,
cDNA cloned into the pRc/CMV
expression vector (21) was transfected into K562 cells by
electroporation, and then 18 days after transfection, G418-resistant
colonies were selected, and expression of
was
verified by flow cytometry. The K562-A3C0 transfectant contained
cDNA with the cytoplasmic tail region terminated
after GFFKR, and K562-A2C3 cells contained
cDNA in
which the tail of
replaced the
tail.
(
)The P388D1 mouse macrophage cell
line and the transfected variant P388D1-
(22) were maintained in RPMI 1640 media with 15% FBS.
Chinese hamster ovary cells (CHO) and variants CHO-A2(23) ,
CHO-A3(20) , and CHO-B1 (24) transfected with human
,
, and
cDNAs,
respectively, were grown in
-minus minimum essential media (Life
Technologies, Inc.) supplemented with 10% FBS. The CHO-A3B1 cell line
(provided by Dr. J. Weitzman) expressing both human
and
subunits, was similarly maintained, except
that 2 mg/ml G418 was added to the growth medium. A mutant strain of
CHO cells transfected with human
cDNA (25) (CHOB2a27 is called CHOB2-A5 in this paper) was obtained
from Dr. R. Juliano. The human breast carcinoma MDA-MB-231 and mammary
epithelial MTSV 1-7 cell lines were cultured in DMEM supplemented
with 10% FBS, with the latter additionally supplemented with 5
µg/ml hydrocortisone (Sigma) and 10 µg/ml insulin (Sigma) as
previously described(26) .
Antibodies
Anti-integrin mAbs used were
anti-, A2-2E10, and A2-3E6(27) ;
anti-
, IA3(28) ; P1B5(29) ;
A3-IIF5(20) ; anti-
, A5-PUJ2(6) ;
anti-
, A6-ELE(6) ; and anti-
,
A-1A5(30) . Also, we used anti-CD63 mAbs RUU-SP
2.28(31) , AHN-16.1 (From M. Skubitz, University of Minnesota),
and the negative control mAb P3(32) . The anti-CD109 mAb 8E11
was generated as previously described(33) . Rabbit polyclonal
serum against the cytoplasmic domains of the integrin
(34) ,
(35) ,
(36) (serum 161-C2), and
(36) (serum 363) subunits were previously described.
Rabbit sera against alternatively spliced variants of the
cytoplasmic domain (6845, anti-
; 382,
anti-
) were generously provided by Dr. V. Quaranta.
Rabbit polyclonal serum against human cathepsin D was purchased from
Sigma.
Antibody Production
RBF/DnJ mice were
immunized four times with MTSV 1-7 mammary epithelial cells
collected in PBS with 5 mM MgCl, and hybridoma
clones were then produced essentially as previously
described(33) , except that the fusion of splenocytes with the
P3X myeloma cell line was done at a 2:1 ratio. After 12 days of
selection in hypoxantihine/aminopterin/thymidine-containing medium,
hybrid clones were analyzed by immunoprecipitation as described below.
Immunofluorescence
For immunofluorescence
studies, HT1080 cells were grown on coverslips for 48 h, fixed for 20
min with 2% formaldehyde in PBS containing 5 mM MgCl and 5% sucrose, permeabilized for 5 min with 0.5% Triton X-100 in
PBS, and then blocked with 10% goat serum in PBS. Cells were stained
with 6H1 mAb (
20 µg/ml in culture supernatant) or with RUU-SP
2.28 mAb (1:200 dilution of ascites in PBS with 10% goat serum) and
subsequently with FITC-conjugated goat anti-mouse serum (Cappel,
Durham, NC). In double-staining experiments, fixed and permeabilized
HT1080 cells were incubated with both anti-CD63 mAb (6H1 or RUU-SP
2.28) and rabbit polyclonal anti-cathepsin D serum (1:500 dilution) and
then developed with both FITC-conjugated goat anti-mouse and
rhodamine-conjugated goat anti-rabbit sera (Cappel). For
CD63/
colocalization experiments, the
6H1 and A3-IIF5 mAbs were directly conjugated with rhodamine and
fluorescein (Pierce), respectively, using the manufacturer's
protocol. HT1080 cells grown on coverslips (for 48 h) were preincubated
in PBS containing 5 mM MgCl
for 15 min, fixed with
2% paraformaldehyde, and then blocked with 10% goat serum/PBS.
``Footprints'' left behind by cells were then stained with
the fluorescent 6H1 and A3-IIF5 mAbs. The coverslips were mounted with
FluorSave Reagent (Calbiochem), and immunofluorescence was examined
using a Zeiss Axioscop equipped with optics for epifluorescence.
Immunoprecipitation
Cells were labeled
with ImmunoPure NHS-LC-Biotin (Pierce) according to the
manufacturer's protocol, and cellular proteins were extracted for
1 h at 4 °C using either mild conditions (25 mM Hepes, pH
7.5, 150 mM NaCl, 5 mM MgCl, 1% Brij 96,
2 mM phenylmethylsulfonyl fluoride (Sigma), 10 µg/ml
aprotinin (Sigma), 10 µg/ml leupeptin (Sigma), 2 mM NaF)
or more stringent conditions (lysis buffer supplemented with 0.2% SDS).
Insoluble material was pelleted at 12,000 rpm for 10 min, and the cell
lysate was incubated with protein A-Sepharose 4CL (Pharmacia Biotech
Inc.) for 1 h to remove background-binding material. The cell extract
was then incubated with specific mAb for 1-2 h at 4 °C, and
the resulting immune complexes were isolated using rabbit anti-mouse
polyclonal antibody (Sigma) preadsorbed onto protein A-Sepharose and
then washed four times with the appropriate immunoprecipitation buffer.
Proteins were eluted from protein A beads using Laemmli sample buffer,
resolved on SDS-PAGE gels, and transferred to nitrocellulose.
Biotinylated proteins were detected by incubation with Extravidin
horseradish peroxidase (Sigma) followed by the Renaissance
chemiluminescent developing reagent (DuPont NEN). For some experiments,
cells were surface labeled with NaI
using
lactoperoxidase/glucose oxidase according a published
protocol(37) , and immunoprecipitation proceeded as described
above. For initial experiments, several other detergents (Nonidet P-40,
SDS, Triton X-100, Digitonin) were substituted for Brij 96 in the
extraction buffer. For metabolic labeling experiments, HT1080 cells (2
10
) were starved for 1 h in methionine-free DMEM
and then labeled with 3 mCi of [
S]methionine for
1.5-2 h. After a chase with complete DMEM, 10% FBS for
3.5-5 h, cells were lysed and immunoprecipitation was carried out
as above.
Reimmunoprecipitation
For reprecipitation
of integrin subunits, HT1080 cells (5
10
-10
) were surface labeled with ImmunoPure
NHS-LC-Biotin, lysed in extraction buffer, and immunoprecipitated
either with 6H1 or RUU-SP 2.28 mAb (anti-CD63). Immune complexes were
collected on protein A-Sepharose beads, washed four times with
extraction buffer, and subsequently eluted from the beads at 70 °C
for 10 min in 0.5 ml of extraction buffer supplemented with 0.5% SDS.
The eluate was diluted 3-fold with extraction buffer and reprecipitated
using specific anti-integrin cytoplasmic domain polyclonal antibodies
that were preadsorbed onto protein A beads. In positive control
experiments, protein lysate prepared in extraction buffer containing
SDS was heat treated in identical fashion but then directly
immunoprecipitated with anti-integrin polyclonal sera.
10
) were metabolically labeled with 5 mCi
[
S]methionine in a methionine-free DMEM for 1.5
h and chased in a complete DMEM, 10% FBS for 4-5 h. After lysis
in 1% Brij 96-containing buffer, the
and
immune complexes were
recovered on Sepharose 4B beads directly conjugated to anti-
(A2-IIE10) and anti-
(A3-IVA5) mAbs,
respectively. The integrin-associated proteins were eluted from the
immune complexes in lysis buffer additionally supplemented with 0.2%
SDS at 4 °C for 30 min and subsequently reprecipitated with 6H1
mAb. The reprecipitated material was resolved on SDS-PAGE and analyzed
as above.
Purification and Sequencing of 6H1
Antigen
From K562 cells (30 g), lysate was prepared in 100
ml of buffer containing 1% Nonidet P-40, 20 mM Tris-HCl, pH
7.5, 0.15 M NaCl, 2 mM phenylmethylsulfonyl fluoride,
5 µg/ml leupeptin, and 5 µg/ml aprotinin. To remove background
bead-binding material, the lysate was sequentially preincubated with
protein A-Sepharose (for 5 h at 4 °C) and Sepharose beads
conjugated with irrelevant mAb (for an additional 4 h). To isolate the
6H1 antigen, the lysate was incubated for 4 h in batch with immobilized
6H1 mAb, coupled at 3 mg/ml packed Sepharose 4B beads (Pharmacia
Biotech Inc.). After washing the beads with 50 column volumes of lysis
buffer, the 6H1 antigen was eluted from the beads using 0.1 M glycine (pH 2.7), and the fractions were immediately neutralized
with 0.1 volume of 1 M Tris-HCl (pH 8.8). Eluted fractions
were analyzed by SDS-PAGE (8% acrylamide), followed by silver staining.
Larger quantities of the fractions containing the 6H1 antigen were then
subjected to SDS-PAGE, and proteins were transferred to polyvinylidene
difluoride membrane (Bio-Rad). The major protein band was visualized by
Ponceau S staining, excised from the membrane, and then amino-terminal
sequencing was carried out using an Applied Biosystems 470A gas-phase
sequenator equipped with a 120A phenylhydantoin amino acid analyzer
(Harvard microsequencing facility, Cambridge, MA). The resulting
amino-terminal sequence was compared to published sequences available
in the GenBank data base using the FASTA program.Flow Cytometry
Cells were harvested,
washed once with PBS containing 5 mM MgCl, and
preincubated in WB5 buffer (PBS containing 5% bovine serum albumin,
0.1% fetal calf serum, and 0.02% sodium azide) at 4 °C for
10-15 min. Typically, 5
10
cells in 0.1 ml
were incubated either with an equal volume of culture supernatant or
with 1:100 dilution of ascites or with 20 µg/ml of purified primary
mAb at 4 °C for 45 min followed by staining with FITC-conjugated
goat anti-mouse secondary Ab (1:100 dilution in WB5 buffer). In
experiments testing for possible cross-reactivity of the 6H1 mAb with
integrins, cells were incubated with
100 µg of the purified
primary antibody (well above the saturation level). Fluorescent
staining was analyzed using a Becton Dickinson FACScan device.
Identification of Integrin-associated
Proteins
To search for integrin-associated molecules, we
chose immunoprecipitation as our major approach. After testing many
different combinations of various detergents, we found that buffer
containing 1% Brij 96 most consistently immunoprecipitated integrins together with putative associated molecules and with
relatively low background. Also, we found that cell-surface labeling
with biotin was more informative than either labeling with
I-labeled iodine or metabolic labeling with
[
S]methionine. With this background information,
we then immunized mice with intact MTSV 1-7 mammary epithelial
cells and attempted to select mAbs against putative
integrin-associated proteins.
and
subunits. To select against clones secreting antibodies that
directly recognize integrins, flow cytometry was carried out using
transfected cells expressing various integrin
and
subunits. Two new mAbs bound to K562-A2 but not K562 cells, six
recognized K562-A3 but not K562, one bound to CHOB2-A5 but not CHO, two
bound to K562-A6 but not K562, and one recognized CHO-B1 but not CHO.
Because these were likely to be anti-integrin antibodies, they were not
considered further. From the remaining antibodies, the mAb secreted by
the hybridoma clone 6H1 was analyzed in more detail.
(lanec) but
distinct from bands coprecipitated along with other integrins (lanesb, d, and e). Other protein
bands in lanea are probably background bands since
they also appeared in the negative control lane (lanef). However, as discussed below, the antigen directly
recognized by 6H1 is not shown in this figure since it is not surface
labeled by biotin.
,
,
or
integrin subunits (not shown). Accordingly, the
6H1 mAb at a concentration of 100 µg/ml did not recognize CHO-A2,
CHO-A3, CHOB2-A5, P388-A6, or CHO-B1 transfectants (Fig. 2, middlecolumn), indicating that the antibody did not
directly recognize the human integrin
,
,
,
, or
subunits expressed on either mouse (P388D1) or hamster (CHO)
cells. The rightcolumn in Fig. 2confirms
that each transfected integrin subunit is expressed at levels
substantially higher than the negative controls (leftcolumn). To exclude the possibility that the 6H1 mAb may
recognize a combinatorial epitope formed by the coexpression of both
human
and
integrin subunits, an
additional flow cytometry experiment was carried out. CHO cells
transfected with both
and
subunits
could not be stained with the 6H1 mAb even at a concentration as high
as 150 µg/ml (not shown), confirming that this mAb was not
cross-reacting with
integrin.
, A2-2E10; anti-
, A3-IVA5;
anti-
, A5-PUJ2; anti-
, A6-ELE;
anti-
, A-1A5. All antibodies were used at
20
µg/ml, except for 6H1, which was used at 100
µg/ml.
integrins (38) , again indicating that
integrins are not required for the cellular reactivity of this
antibody.
and
subunits, a reprecipitation experiment was carried out (Fig. 3). Following SDS disruption of a 6H1 immune complex
isolated from biotin-labeled HT1080 cells, proteins of 125-130
kDa could be reprecipitated using polyclonal antibodies against the
cytoplasmic domains of
,
, and
(lanesj, l, and n). In contrast, neither
nor
integrin subunits were reprecipitated from the 6H1 mAb immune
complex (lanesi and k). Positive control
experiments showed that anti-
, -
,
-
, -
, -
, and
-
sera were each fully capable of recognizing the
appropriate integrins from HT1080 cell lysates (Fig. 3, lanesa-f). The
subunit,
expressed at only a low level on HT1080 cells, also could be
reprecipitated from the 6H1 immunoprecipitate (data not shown). In two
additional cell lines (MTSV 1-7, MDA-MB-231), the 6H1 mAb again
coprecipitated the
1 integrin as determined by
reprecipitation experiments (not shown). The 6H1 protein itself was
again not visible because it is not biotin labeled (see below).
and
integrins from a 6H1 immunoprecipitate. HT1080 cells were surface
labeled with biotin, lysed in 1% Brij 96 buffer, and then
immunoprecipitated with the 6H1 mAb (laneh).
Material precipitated by mAb 6H1 was eluted from protein A beads in 1%
Brij 96 buffer supplemented with 0.2% SDS at 70 °C and then
reprecipitated with specific polyclonal sera against the indicated
cytoplasmic domains (lanesi-m) or rabbit
preimmune sera (laneo). In a control experiment (to
ensure that all anti-cytoplasmic domain sera were active),
surface-labeled HT1080 cells were lysed in buffer containing 1% Brij 96
and 0.2% SDS, preheated for 10 min at 70 °C, and directly
precipitated with specific polyclonal sera against the indicated
integrin cytoplasmic domains (lanesa-f) or
with preimmune sera (laneg). Precipitates resolved
in 10% SDS-PAGE under reduced conditions.
and
integrins was additionally confirmed in direct
immunoprecipitation experiments, using extracts prepared from
biotin-labeled K562, K562-A2, K562-A3, and K562-A6 cells (Fig. 4). From all four cell lines, the 6H1 mAb consistently
precipitated several proteins of 67, 95, and 110-115 kDa.
However, bands in the region of integrin
subunits (140 and 150
kDa) were detected in immunoprecipitates from K562-A3 and K562-A6 cells (Fig. 4, lanesf and h, respectively)
but not from K562 or K562-A2 cells (lanesd and e). Taken together, these data suggest that the antigen
recognized by the 6H1 mAb could selectively associate with the
and
integrins but not the
or
integrins.
mAb A5-PUJ2 (lanea), anti-a
mAb
A2-2E10 (laneb), anti-a
mAb
A3-IVA5 (lanec), or anti-a
mAb A6-ELE (laneg). Precipitates were eluted into Laemmli
loading buffer and resolved in 10% SDS-PAGE under non-reduced
conditions. The negative control mAb P3 did not precipitate labeled
material from any of the cell extracts used (not
shown).
The 6H1 mAb Recognizes CD63
To
characterize the 6H1 antigen further, HT1080 cells were metabolically
labeled with [S]methionine, and then
immunoprecipitation was performed under stringent conditions (with 0.2%
SDS included in extraction and washing buffers). For the first time, we
could see that material precipitated by mAb 6H1 appeared as a single
broad band ranging from 45 to 60 kDa (Fig. 5, laned). This band was not precipitated by a control antibody (lanef). Notably, when mild conditions were
utilized, 6H1 immunoprecipitation yielded other
S-labeled
proteins, including proteins of 150 and 130 kDa that are likely to be
integrin
,
, and
subunits (Fig. 5, lanec). The prominent
protein of 45-60 kDa seen with
S labeling (lanesc and d) was not obvious upon
I-labeled iodine (not shown) or biotin labeling (lanesa and b). Nonetheless,
130-150-kDa integrin proteins were coprecipitated under mild
conditions (lanea), consistent with these being
indirectly associated with the 6H1 antigen.
S]methionine (lanesc-h). Lysis buffer contained 1% Brij 96 (lanesa, c, e, and g) or 1% Brij 96
and 0.2% SDS (lanesb, d, f, and h). Lysates were immunoprecipitated with mAb 6H1, the
established anti-CD63 mAb RUU-SP 2.28, or the negative control mAb P3
as indicated. Precipitates were resolved in 9% SDS-PAGE under
non-reduced conditions.
S-labeled proteins (Fig. 5, lanesc and g) that were not seen in control lanes (lanese and h). Also, even though the 45-60-kDa
CD63 protein itself was not labeled with biotin, nearly identical
patterns of biotin-labeled proteins were coprecipitated by both the 6H1
and RUU-SP 2.28 mAbs (Fig. 7, lanesb and c). Additional reprecipitation experiments confirmed that the
RUU-SP 2.28 mAb, like 6H1, could coprecipitate
and
integrins from a Brij-96 HT1080 cell lysate (data not shown).
Notably, the proteins coprecipitated by these two antibodies closely
resemble a subset of those proteins coprecipitated by the
anti-
antibody (Fig. 7, lanea). Furthermore, immunofluorescent staining of HT1080
cells showed that the antigenic determinants recognized by both the 6H1
and RUU-SP 2.28 mAbs were not only present on the outer cell membrane
of HT1080 cells but also were highly abundant in perinuclear granules
where they colocalized with cathepsin D (not shown).
mAb P1B5 (lanea), 6H1 mAb (laneb), or RUU-SP 2.28 mAb (lanec). Precipitates were resolved in 9% SDS-PAGE under
non-reduced conditions.
integrins,
we carried out immunoprecipitations of metabolically labeled
and
from HT1080 cells and then looked for the presence of CD63. The
reprecipitation experiment showed that a protein band corresponding to
CD63 was detected in the
immunoprecipitate (Fig. 8, laneb) that
represents 2-5% of the total CD63 directly immunoprecipitated in lanea. No CD63 was detected in an
immunoprecipitate (lanec), even though
is
highly expressed on HT1080 cells (e.g. see Fig. 3). In
addition, we looked for possible cellular colocalization of CD63 with
the
integrin (Fig. 9). Within
HT1080 cells that were fixed but not permeabilized, staining for
integrin
showed abundant distribution throughout the
membrane, with some punctate staining (panelA). The
CD63 molecule was also present through the cell membrane, but the
punctate expression pattern was more obvious (panelB). Colocalization of
integrin (panelC) and CD63 (panelD) was
most obvious in cellular footprints left following the retraction of
spread cells on a coverslip. In a control experiment, another abundant
protein (CD109) was not localized into the same footprints, and neither
was the VLA-2 integrin (not shown). From the above results, we conclude
that the 6H1 mAb recognizes the CD63 protein (39) and that
this protein may associate with
integrin within intact cells as well as in cell lysates.
immunoprecipitate. Integrin immune
complexes were prepared from
S-labeled HT1080 cells using
A2-2E10 (anti-
) and A3-IVA5
(anti-
) mAbs directly coupled to Sepharose 4B beads.
Integrin-associated molecules were eluted from the beads with 0.2% SDS,
reprecipitated with an anti-CD63 mAb (6H1), and resolved on 11%
SDS-PAGE (lanesb and c). A positive control
experiment shows the broad CD63 protein (45-60 kDa) specifically
precipitated by mAb 6H1 (lanea).
integrin in cell footprints. HT1080
cells were grown overnight on coverslips, fixed with 2%
paraformaldehyde, stained with anti-
mAb A3-IVA5 (panelA) and 6H1 anti-CD63 (panelB), and visualized using FITC-conjugated goat anti-mouse
Ig. Also, cell footprints were prepared and stained as described under
``Materials and Methods,'' and double staining was carried
out using FITC-conjugated anti-
A3-IVA5 (panelC) and rhodamine-conjugated 6H1 mAb (panelD). The arrows indicate colocalization of CD63
and
integrin.
The
Because integrin Cytoplasmic Domain Is
Not Required for CD63 Association
subunit cytoplasmic domains have diverse functional
roles(5, 40) , we hypothesized that they might also
mediate selective biochemical associations with proteins such as CD63.
To ascertain whether the cytoplasmic tail of
is
required for the association of the
integrin with CD63, we analyzed K562-A3C0 cells (K562 cells
expressing the
subunit with no cytoplasmic domain).
As shown in Fig. 10, both wild type
(laneb) and the
deletion mutant (laned) could be coprecipitated by the 6H1 mAb from
biotinylated cell extracts. In each case, similar patterns of
additional coprecipitated bands of 67 and 97 kDa were present, and
these closely resembled proteins coprecipitated by the anti-
mAb (lanesa and c). In a separate
experiment, we analyzed K562-A2C3 cells (K562 cells expressing the
chimera, which contains the transmembrane and extracellular domains of
and the cytoplasmic domain of
). As
shown (Fig. 10, lanef), the 6H1 mAb failed to
coprecipitate the chimeric A2C3 or
integrin subunits.
Together, these results suggest that the cytoplasmic domain of the
subunit is neither required nor sufficient for the
formation of the
-CD63 complex.
cytoplasmic
domain in CD63 association. K562-A3, K562-A3C0, and K562-A2C3 cells
were surface labeled with biotin, lysed in 1% Brij 96 buffer, and
immunoprecipitated using mAb 6H1 (lanesb, d, and f), anti-
mAb P1B5 (lanesa and c), or anti-
mAb
A2-2E10 (lanee). Precipitates were resolved in
10% SDS-PAGE under non-reduced conditions. The negative control mAb P3
did not yield labeled material from the cell lines used (not
shown).
integrins with other proteins have not previously been described.
Now, our reciprocal immunoprecipitation data and cellular
colocalization data provide strong evidence for specific
CD63-
and
CD63-
associations.
and
in stronger detergent conditions, consistent with an indirect
reactivity. Second, we have utilized three different anti-CD63
monoclonal antibodies to detect CD63 association with integrins.
Furthermore, high levels of the anti-CD63 mAb 6H1 (well above
saturating) failed to recognize CHO cells transfected with human
,
, or
integrin
subunits, and conversely, no
integrins could be
detected on a cell line (JY) that expresses CD63.
and
but not
or
, even though the latter
were present at similar levels. Second, reciprocal experiments showed
not only that VLA-3 (and VLA-6) were present in CD63 immunoprecipitates
but also that CD63 was present in VLA-3 (but not VLA-2)
immunoprecipitates. Third, both the CD63 and VLA-3 proteins are
colocalized in cellular footprints, as seen by immunofluorescent
staining.
and
integrins and showed a similar
pattern of other coprecipitated proteins on SDS-PAGE and 2) showed
similar profiles of cell-surface staining and localization to cathepsin
D-containing intracellular granules. The latter data are in agreement
with previous reports describing a dual subcellular localization of
CD63(39, 41) .
and
integrins is of interest because
these integrins have some degree of functional
overlap(42, 43, 44, 45) , and their
primary structures are more similar to each other (37%) than to most
other integrin
subunits (46, 47, 48) .
Thus, regions specifically shared between
and
are likely to be critical for their association with
CD63. Because distinct integrin
subunit cytoplasmic domains
sometimes exhibit diverse functions during both inside-out (40) and outside-in (5, 8) signaling events,
we thought that the
and
cytoplasmic
domains might play key roles in CD63 association. However, the integrin
cytoplasmic domain was neither necessary nor
sufficient for CD63 association, and
-CD63 association occurred regardless
of whether the
or
cytoplasmic
domain was present. Thus, it appears that extracellular (or
transmembrane) regions, rather than cytoplasmic domains, must be
critical for CD63 association. Also, the integrin
subunit might
influence CD63 interactions. In preliminary experiments, anti-CD63 mAb
failed to coprecipitate the
subunit from cells where
it was predominantly paired with the
subunit, even
though an
-CD63 complex was readily
detectable in the same cells.
and
showed that both were evenly distributed on the cell surface.
Colocalization of CD63 and
in cell
footprints suggests that the cell-surface fraction of the CD63 antigen
may be more likely to associate with integrins. However, we cannot
exclude intracellular association because it is possible that
conditions used for cell permeabilization might not be optimal for
detection of integrins in intracellular granules. The CD63 protein
and/or mRNA is present in many tissues(55) , with high
expression in the kidney, especially in the
glomerulus(56, 57) . The
integrin is also expressed in kidney glomeruli(58) , and
both
and
are widely expressed on
many cell and tissue types (59, 60, 61, 62) , as well as on
nearly all cultured adherent cell lines. Consistent with this, we found
that
-CD63 and
-CD63 complexes could be detected in
cells of different tissue origins (lymphoid, mesenchymal, epithelial).
Thus, the possibility exists that integrin-CD63 association could be
widespread and of general importance for cellular physiology.
and
CD63-
associations. Nonetheless, we
hypothesize that such associations might specifically affect signaling
through
and
integrins and could be important for
cell proliferation, migration, or cell-cell adhesion. The
colocalization of CD63 with
integrin
in footprints left by cells on the different extracellular matrix
substrates ( Fig. 9and preliminary results) suggests that the
function(s) of the complexes could be related to the adhesive
properties of integrins. In this regard, CD63 and other members of the
TM4 protein family may modulate cell proliferation(63) , cell
motility (64) , and cell-cell
adhesion(31, 65) . Likewise, various
integrins (including
) play key
roles in regulating cell proliferation(24, 66) , cell
migration(8, 67) , and cell-cell
adhesion(68, 69) .
(
)Another
possibility is that CD63 association with
and
might modulate the
recycling of these integrins. Notably, it has been reported that
has a reduced rate of endocytic
recycling compared to
and
integrins(70) .
(e.g.Fig. 7). These results suggest that
association of
and
integrins with CD63 could possibly
occur within the context of a larger multiprotein complex so that
instead of a direct interaction, these proteins are connected with one
another via other proteins present in the complex. In this respect, our
preliminary experiments show that other members of the TM4 family can
interact with both CD63 and
/
integrins. Also, it is interesting to note that another member of
the TM4 family, the CD9 antigen, was suggested to associate with the
integrin
on platelets (71) and with VLA-4 and VLA-5 integrins on leukocyte cell
lines(72) .
integrins and highly reproducible in many experiments
from multiple cell types. Awareness of these
CD63-
and
CD63-
interactions now provides the
opportunity to gain new insights into the functional properties of
,
,
and CD63.
We thank Drs. R. Juliano and A. Mercurio for
integrin-transfected cell lines; Drs. John McDonald, V. Quaranta, and
R. Hynes for polyclonal antisera; A. Mercurio for cDNA; Drs. K. Skubitz, K. Nieuwenhuis, and E. Wayner for
monoclonal antibodies; and R. Pasqualini for help during the initial
stages of hybridoma production.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.