(Received for publication, August 17, 1995)
From the
The cytoplasmic domains of integrin subunits are involved
in bidirectional transmembrane signaling. We report that the
cytoplasmic domain of the integrin
subunit undergoes
limited proteolysis by calpain, an intracellular calcium-dependent
protease. Calpain cleavage occurs during platelet aggregation induced
by agonists such as thrombin. Five cleavage sites have been identified.
Four of these sites (C-terminal to Thr
,
Tyr
, Phe
, and Tyr
) are
utilized in intact platelets and flank two NXXY motifs
(Asn
-Pro-Leu-Tyr
and
Asn
-Ile-Thr-Tyr
). The fifth site
(Ala
) is accessible to calpain after EDTA treatment of
the
heterodimer. The NXXY
motif is critical to the bidirectional signaling functions of
integrins and their association with the
cytoskeleton. Thus, calpain cleavage of the
cytoplasmic domain may provide a means to regulate integrin
signaling functions.
Integrins, a family of adhesion receptors, play important roles
in cellular functions such as adhesion, migration, cell proliferation,
and differentiation(1) . The functions of integrins are
modulated by bidirectional transmembrane signaling as exemplified by
platelet integrin (glycoprotein
IIb-IIIa)(2) . When platelets are stimulated by agonists such
as thrombin, intracellular signal transduction leads to activation of
the extracellular fibrinogen binding function of
(inside-out signaling), resulting
in platelet aggregation. Conversely, ligand binding to
is involved in outside-in signals
that result in its association with the cytoskeleton (3) and
biochemical changes including tyrosine phosphorylation of intracellular
proteins(4, 5) , increase in intracellular calcium
level(6) , and activation of the calcium-dependent neutral
protease (calpain)(7) . Specific structural characteristics of
the integrin cytoplasmic domains are critical for the two-way signaling
mechanism. This includes a GFFKR sequence in the
cytoplasmic domain (8) and two sets of NXXY
(NPLY and NITY) within the
cytoplasmic
domain(9, 10) . The NPLY sequence, in particular, is
critical for controlling affinity states of the ligand-binding sites
and for the interaction of the integrin with cytoskeletal
elements(9, 10) .
Numerous cytoplasmic proteins are
colocalized with integrins in focal adhesion sites and thus may be
involved in intracellular signaling to and from integrins(11) .
Calpain is among these proteins(12) . Calpain represents a
family of intracellular calcium-dependent neutral
proteases(13) . Of the two classic members of this family,
µ-calpain and m-calpain, µ-calpain is probably the predominant
form in platelets(14) . These two isoforms of calpain have no
major difference in substrate specificity, but differ in calcium
sensitivity (14) . Calpain can be activated during platelet
aggregation by a rise in the cytoplasmic calcium level and/or by its
translocation to the membrane(13, 14) . Furthermore,
calpain activation promotes the shedding of procoagulant membrane
vesicles from aggregated platelets (15) . While the mechanisms
of calpain regulation are still not fully understood, calpain cleavages
can regulate a variety of intracellular
processes(16, 17, 18, 19) . We now
report that calpain may regulate the function of integrins by limited cleavage of the cytoplasmic domain of
at specific sites flanking two sets of NXXY
motifs.
Figure 1:
Cleavage of purified integrin
by µ-calpain. Integrin
was incubated with µ-calpain
at 30 °C for 5 min and then analyzed by SDS-PAGE and Western
blotting. A, control or calpain-treated integrin
was immunoblotted with rabbit
antipeptide antibody anti-
C (against the C-terminal 20
amino acid residues of the
cytoplasmic domain),
antibody 8053 (against the entire
subunit),
monoclonal antibody PMI-1 (against the
heavy
chain), and rabbit antipeptide antibodies anti-IIbC (against the C
terminus of the cytoplasmic domain of the
light
chain) and anti-V41 (against the N terminus of the
light chain). B, shown is a schematic of the
localization of epitopes recognized by the various
anti-
antibodies used in A. MAb, monoclonal
antibody.
In contrast, the cytoplasmic domain of did not
appear to be cleaved by calpain, as indicated by the preservation of
reactivity with an antibody to the C terminus of the
cytoplasmic domain (anti-IIbC). In addition, reactivity with an
antibody to the C terminus of the
heavy chain
(PMI-1) was also unaffected by calpain. The N terminus of the
light chain appeared to be cleaved since a second
more mobile band, reactive with the antibody against the C terminus of
the
light chain, became evident after calpain
treatment (Fig. 1). This more mobile band did not react with
antibody anti-V41 (specific for the N terminus of the
light chain), and anti-V41 staining of the intact
light chain was decreased by calpain treatment. Thus, the
cytoplasmic domain resists calpain cleavage, while
the N terminus of the light chain is susceptible. Consequently, the
cytoplasmic domain of integrin
appears to be a
preferred intracellular substrate for calpain.
To determine if
cleavage of the cytoplasmic domain by calpain occurs
in intact cells, washed platelets were treated with a calcium
ionophore, A23187 (1 µM), for 3 min at 37 °C to
activate intracellular calpain. After solubilization, intact
subunit was depleted by sequential
immunoprecipitation with anti-
C, and residual
was detected by Western blotting with an antibody to
the extracellular domain of
(monoclonal antibody 15).
Densitometry showed that >70% of
from
A23187-treated platelets remained in the supernatant after quantitative
immunoabsorption by anti-
C. This result indicates that
most of the
subunit in A23187-activated platelets
lacks the C-terminal portion. In contrast, >99% of
in resting platelets was depleted by immunoprecipitation with
anti-
C, indicating that the cytoplasmic domain of
is intact (Fig. 2A). These results
were confirmed when platelets were directly solubilized in SDS sample
buffer containing calpain inhibitors and then Western-blotted with
anti-
C. The integrin
subunit from
A23187-stimulated platelets showed much less reactivity with
anti-
C than did
from control
platelets. This reduction in the anti-
C reactivity was
due to calpain cleavage because a membrane-permeable calpain inhibitor,
E-64d, blocked it completely (Fig. 2B). Thus,
calpain-dependent cleavage of the cytoplasmic domain of
occurs in intact platelets.
Figure 2:
Calpain cleavage of in
platelets. A, washed platelets in modified Tyrode's
buffer (0.4 ml) were incubated at 37 °C with (A23187) or without
(control) 1 µM calcium ionophore A23187 for 3 min with
continuous stirring at 1000 rpm. The platelets were solubilized by
addition of an equal volume of solubilization buffer (see
``Experimental Procedures'') and immunoprecipitated twice
with 50 µl of preimmune serum (control (Ctrl)),
anti-
antibody 8053 (
), or
the anti-
C-terminal peptide antibody,
anti-
C (
C). After
removing the antibody-bound proteins with protein A-conjugated
Sepharose beads, the 50 µl of supernatant were fractionated by
SDS-PAGE and immunoblotted with a monoclonal anti-
antibody, 15. B, platelets were stirred at 37 °C for
3 min after adding buffer (0), 1 µM A23187 (A), or A23187 with 0.1 mM E-64d (a
membrane-permeable calpain inhibitor) (A+E) and then
solubilized by addition of an equal volume of SDS sample buffer
containing 0.2 mM E-64, 1 mM PMSF, and 5 mM EDTA. 50 µl of lysates were analyzed by SDS-PAGE and
immunoblotted with preimmune serum, antibody 8053, or
anti-
C.
Figure 3:
Mapping calpain cleavage sites in the
cytoplasmic domain of . A synthetic peptide
(TIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT) corresponding to the
cytoplasmic domain of integrin
(100 µg) was
incubated in the absence (A) or presence (B) of
purified calpain (1 µg) at 30 °C for 20 min. The samples were
analyzed by reverse-phase HPLC using a C
column. The
fragments were eluted with a gradient of 15-60% acetonitrile in
0.1% trifluoroacetic acid. In C, peaks (fragments
a-g) eluted from the HPLC column were immediately analyzed
by ion-spray mass spectrometry, and their sequences were determined as
described under ``Experimental Procedures.'' Calpain cleavage
sites (arrows) were assigned by the location of these
fragments in the intact peptide. The solid circles above the arrows indicate cleavage sites confirmed by N-terminal
sequencing in a separate experiment.
Three of the cleavage site assignments were
confirmed by N-terminal sequence analysis of the products of calpain
digestion of the cytoplasmic domain peptide,
Glu
-Thr
. Three HPLC peaks were found
to contain peptides with N-terminal sequences differing from the intact
peptide: A
NNPLYKEA . . . , K
EATSTF . . . ,
and T
NITYRGT, confirming three of the calpain cleavage
sites identified by mass spectrometry (Fig. 3C). Thus,
five calpain cleavage sites were identified C-terminal to
Ala
, Thr
, Tyr
,
Phe
, and Tyr
of the
subunit.
Figure 4:
Calpain cleavage site utilization in
integrin in intact platelets. Washed platelets
resuspended in Tyrode's buffer (10
/ml) were treated
with 1 µM A23187 in the absence (A) or presence (A+E) of 1 mM E-64d. Alternatively, no A23187
was added (0). After 3 min at 37 °C, platelets were
solubilized by addition of an equal volume of SDS sample buffer
containing 0.2 mM E-64, 0.5 mM leupeptin, 1 mM PMSF, and 5 mM EDTA. The proteins were separated by
SDS-PAGE and then immunoblotted with the indicated calpain cleavage
site-specific antibodies. Antibody binding was visualized by
peroxidase-conjugated goat anti-rabbit antibodies and
ECL.
In contrast,
the site C-terminal to residue 735 did not appear to be cleaved by
calpain in the cellular environment. To verify that Ab 735 recognizes
appropriately cleaved , platelets were treated with 10
mM EDTA at 37 °C for 1 h to dissociate
from
. After solubilization, 40 mM CaCl
was added to the lysate to activate endogenous
calpain. After such treatment, reactivity of Ab 735 with
was detected (Fig. 5), indicating that cleavage after
Ala
had occurred. An upward shift in mobility of
on a nonreduced SDS-polyacrylamide gel, similar to
that of reduced
, was also observed. This suggests
that cleavage may occur in the extracellular domain, leading to the
unfolding of an extracellular loop within
(29) . Thus, the calpain cleavage site
C-terminal to Ala
is utilized following treatments known
to dissociate the
and
subunits.
Figure 5:
Calpain
cleavage of at Ala
in EDTA-treated
platelet lysate. Washed platelets were treated with 10 mM EDTA
(+) or incubated in the presence of 2 mM CaCl
(-) at 37 °C for 30 min and then
solubilized by adding an equal volume of 2% Triton X-100, 0.1 M Tris, 0.15 M NaCl, pH 7.4, containing 1 mM PMSF,
but no calpain inhibitors. After an additional incubation at 37 °C
for 60 min, a final concentration of 40 mM CaCl
was added to EDTA-treated platelet lysates to activate calpain.
The lysates were incubated at room temperature for a further 30 min and
then fractionated by SDS-PAGE (nonreduced) and immunoblotted with Ab
735 or Ab 754. Note that the Ab 735 epitope appeared only when the
platelets were pretreated with EDTA before calpain
activation.
Figure 6:
Calpain cleavage of integrin in platelets activated by thrombin. Washed platelets
(10
/ml, 0.4 ml) were treated at 37 °C in the absence (0) or presence of 0.1 unit/ml thrombin for 1, 3, 5, or 10 min
with continuous stirring at 1000 rpm. The reactions were stopped by
adding an equal volume of SDS sample buffer containing 0.2 mM E-64, 0.5 mM leupeptin, 2 mM PMSF, and 5 mM EDTA. Proteins in the platelet lysates were separated by SDS-PAGE
and then immunoblotted with the calpain cleavage site-specific antibody
(Ab 754).
We have found that the cytoplasmic domain of the integrin
subunit is cleaved by calpain and have mapped five
cleavage sites. Four of these sites (C-terminal to Thr
,
Tyr
, Phe
, and Tyr
) are
utilized in intact platelets. The fifth site is accessible only after
treatment known to dissociate the
heterodimer. These calpain cleavages remove residues critical for
the attachment of the integrin to the cytoskeleton and bidirectional
transmembrane signaling. Thus, calpain cleavage may regulate functions
of
integrins.
The conclusion that calpain cleaves
the cytoplasmic domain of integrin comes from three
lines of evidence: 1) in vitro cleavage of the
cytoplasmic domain of purified integrin
by purified µ-calpain, 2)
cleavage of synthetic
cytoplasmic domain peptides by
purified µ-calpain, and 3) limited cleavage of the cytoplasmic
domain of the integrin in intact platelets stimulated by A23187 or
thrombin. Although the conditions differ significantly, the results
from these experiments are highly consistent, indicating that primary
sequence-defined structures of the
cytoplasmic domain
are recognized by calpain. Calpain cleavages released only small
peptide fragments from the C-terminal region of
,
resulting in no significant shift in its mobility on SDS-PAGE. This may
explain why calpain cleavage of the
subunit was not
noted previously(30) .
Calpain cleavage of occurs during platelet aggregation, suggesting that it may
regulate platelet function. Immunoabsorption with an antibody specific
for the
C terminus almost completely depleted
from resting platelets. In comparison, >70% of
in A23187-activated platelets did not react with this
antibody (Fig. 2A). This indicates that the bulk of
in resting platelets is intact. However, calpain
cleavage of
was observed as early as 1 min after
adding a physiological platelet agonist (thrombin) and increased with a
time course similar to that of calpain activation in thrombin-activated
platelets (Fig. 2B)(7) . Platelet aggregation
leads to calpain cleavage of cytoskeletal proteins such as talin (16) and signaling molecules such as protein-tyrosine
phosphatase IB(18) , pp60
(17) , and
protein kinase C(19) . Interestingly, the
subunit
cytoplasmic domain is believed to interact with
talin(31, 32) , and integrins regulate the functions
of the other signaling
molecules(11, 17, 18) . Thus, it is possible
that calpain cleavage of both the cytoplasmic domain of an integrin and
its downstream signaling partners may be a coordinated process. In
vitro digestion of the purified integrin by calpain also
established cleavage at the N-terminal domain of the
light chain, which is consistent with an earlier observation that
may be proteolytically modified(24) . This
cleavage is not regulated by platelet activation(24) .
Calpain cleavage site utilization in intact cells was detected with
antipeptide antibodies specific for the cleavage sites. Each antigenic
peptide had a 5-residue sequence (normally a minimum required for an
epitope) corresponding to the C terminus generated by a predicted
calpain cleavage. The antipeptide antibodies reacted preferentially
with calpain-cleaved (Fig. 4). As described
previously (26) and in this study (Fig. 4), this
strategy can thus be used to identify protease cleavage site
utilization in vivo.
The subunit may
affect calpain access to cleavage sites in the
cytoplasmic domain. Four of five calpain cleavage sites in
were cleaved by calpain in intact platelets. However,
the most membrane-proximal cleavage site (Ala
) became
susceptible to calpain cleavage only after pretreatment of the integrin
with EDTA. As EDTA is known to dissociate the calcium-dependent complex
of
and
subunits(28) ,
this result suggests that the region near Ala
was
shielded from calpain cleavage in the complexed
heterodimer. Thus, either the
interaction of
with
may regulate
the conformation of the
cytoplasmic domain, or the
cytoplasmic domain of
may directly interact with
the cytoplasmic domain of
at a site close to
Ala
.
Calpain specificity is not defined solely by the
amino acid residues flanking the scissile bonds(14) . Thus, the
secondary and tertiary structures of the protein in the vicinity of the
scissile bond may be important determinants of cleavage.
Peptide-derived calpain inhibitors such as chloromethyl ketones
(Leu-Leu-Tyr-CHCl and Leu-Leu-Phe-CH
Cl) contain
the hydrophobic residues (leucine) N-terminal to an aromatic amino acid
residue (Tyr or Phe)(33) . Moreover, a hydrophobic residue
(corresponding to P2 and P3 positions) N-terminal to an aromatic or a
positively charged residue at the cleavage site is a pattern frequently
present in calpain substrates (14) . The four calpain cleavage
sites in the
cytoplasmic domain identified in intact
platelets (
A
NNPLY
and
T
NITY
) flank two NXXY
motifs. The two NXXY motifs in the
cytoplasmic domain each contain a leucine or isoleucine at one of
the X positions, N-terminal to a tyrosine at the calpain
cleavage site. Thus, the NXXY motifs are similar to cleavage
sites found in other calpain substrates. Calpain cleavage also occurs
on the N-terminal side of the NXXY sequence. In the low
density lipoprotein receptor, the NPXY motif forms a tight
turn so that both N- and C-terminal flanks are sterically
adjacent(34) . It is possible that such turns could exist in
the
cytoplasmic domain.
Cleavage of the
cytoplasmic domain by calpain near two NXXY
sites may be an important mechanism for the regulation of its
bidirectional signaling and
attachment to the cytoskeleton following ligand binding. The more
N-terminal NXXY motif of the
cytoplasmic
domain has the sequence NPLY, similar to the NPXY
internalization signal identified in the low density lipoprotein
receptor(35) . Mutations disrupting this motif abolish the
capacity of the
cytoplasmic domain to regulate the
affinity state of the receptor (inside-out signaling)(9) , to
associate with the cytoskeleton at focal adhesion
sites(10, 36) , and to mediate cell
migration(37) . Furthermore, NXXY motifs containing
hydrophobic residues at one of the X residues are highly
conserved in the cytoplasmic domain of most integrin
subunits,
including
,
,
,
,
, and
, and have
been implicated in the functions of some of these
subunits(38, 39) . Thus, it is possible that calpain
cleavage may also occur to these integrins near the NXXY motif
and regulate the functions of these integrins. So far, only the
cytoplasmic domain has been reported to be a
substrate for calpain(40, 41) . The
cytoplasmic domain is very different from that of other integrin
subunits in primary sequence, size (1019 residues), and
function(42, 43) . Although the
cytoplasmic domain also contains an NXXY
sequence(42) , calpain cleavage sites in the
cytoplasmic domain have not been accurately identified and thus
cannot be compared with cleavage sites in
as
described in this study.
The work presented here provides the first
evidence of physiologically regulated calpain cleavage of an integrin
cytoplasmic domain. In platelets, cleavage of the subunit could limit or reverse platelet aggregation and could
permit relaxation of contracted fibrin clots. It may also be important
to the calpain-dependent shedding of integrin-containing, procoagulant
membrane vesicles during platelet aggregation(44) . As calpain
colocalizes with integrins in focal adhesion sites, calpain cleavage at
these sites may also serve as a mechanism to detach migrating cells
from the extracellular matrix while leaving an integrin
``trail'' behind(45) .