(Received for publication, May 15, 1995; and in revised form, June 21, 1995)
From the
We have previously demonstrated an interaction between the
highly conserved KXGFFKR sequence of the integrin
-subunit cytoplasmic domains and calreticulin. Since this highly
conserved sequence motif has been implicated in the regulation of the
integrin affinity state, we wanted to determine whether the
calreticulin-integrin interaction also depended on the integrin
affinity state, and whether calreticulin occupation of integrin via the
KXGFFKR motif was involved in the regulation of the ligand
affinity state. We now demonstrate that anti-integrin antibody- or
phorbol 12-myristate 13-acetate (PMA)-induced activation of the
integrin on Jurkat cells, as
determined by stimulation of adhesion to collagen type I, resulted in
an increased amount of calreticulin bound to this integrin.
activation with either
anti-
or anti-
monoclonal antibodies
resulted in a greater amount of calreticulin coimmunoprecipitating with
this integrin. Inactivation by neutralizing anti-integrin antibodies
abrogated the calreticulin-integrin interaction. A correlation was also
found between PMA-induced
activation
and the amount of calreticulin bound to this integrin. Furthermore,
pretreatment of streptolysin O-permeablized Jurkat cells with an
anti-calreticulin antibody resulted in a significant and specific
inhibition of the adhesion to collagen type I that could be induced by
antibodies to
or by PMA. These data
suggest that the active, high affinity form of
binds calreticulin and that
calreticulin binding to the
cytoplasmic domain may be
required for stabilizing the high affinity state of this integrin. The
data presented here also demonstrate, for the first time, an inducible
interaction of an integrin with an intracellular protein that occurs
via the
subunit of the integrin.
The integrins are a family of heterodimeric, transmembrane
glycoproteins that are formed by the noncovalent association of
and
subunits(1, 2) . As transmembrane proteins,
integrins can interact with both extracellular molecules and
intracellular proteins, interactions which define integrin function. On
the cell exterior, domains of the
and
subunits combine to
form a ligand binding site. Through this site, integrins can mediate
cell binding to various substrates including extracellular matrix
molecules(3, 4) , other cell surface
proteins(1, 5) , and in some cases, other
integrins(6) . Interior to the plasma membrane, the cytoplasmic
domains of integrin subunits can engage in interactions with
intracellular proteins(7, 8) , and these interactions,
unlike those of extracellular ligand binding, can occur independently
of subunit association in vitro. The best studied cytoplasmic
interactions in which integrins take part are those involving the
subunit. The cytoplasmic domain of the
subunit has been shown to interact with components of the
actin-based cytoskeleton, namely
-actinin (9) and
talin(10) . In contrast to these
subunit-specific
functions, cytoplasmic interactions involving the
subunits have
not been as well characterized.
The cytoplasmic domains of the
integrin subunits are diverse in both length and amino acid
composition. The only generally conserved feature within the
subunit cytoplasmic domains is the KXGFFKR motif which
immediately follows the membrane spanning
domain(3, 8) . The conservation of this motif in all
subunits suggests that it plays a crucial role in integrin
function. Studies using transfected integrin subunits support this
hypothesis by showing that chimeric integrins containing disrupted
GFFKR motifs are fixed in high affinity ligand-binding
states(11) . Thus the KXGFFKR motif may be important
for maintaining the conformation of the integrin in a low affinity
default state and possibly for additional regulatory functions.
In
what is one of the best examples, to date, of an integrin
subunit-cytoplasmic protein association, the KXGFFKR motif of
integrin
subunits has been shown to interact with the
intracellular protein, calreticulin. Initial experiments revealed that
affinity chromatography of cell extracts using KLGFFKR peptides
resulted in the isolation of a
60-kDa protein with amino acid
sequence homology and immunological similarity to
calreticulin(12) . Subsequently, calreticulin was
immunocytochemically shown to colocalize with integrins, and antisense
oligonucleotide down-regulation of calreticulin led to a decrease in
integrin-mediated cell attachment and spreading(13) .
Calreticulin is a highly conserved, ubiquitously expressed
intracellular protein whose functions are just beginning to be
understood. Calreticulin was first identified as a calcium binding
protein of the sarcoplasmic reticulum in skeletal
muscle(14, 15) . Originally thought to be restricted
to the endoplasmic reticulum, it has subsequently been shown that
calreticulin can localize to the nucleus and/or the cytoplasm in some
cell types(7, 16, 17) . Recently, it has been
shown that calreticulin can bind to steroid hormone receptors and
consequently down-regulate the expression of steroid hormone responsive
genes(18, 19) . Importantly, calreticulin has been
shown to bind to the DNA binding domain of these hormone receptors via
an amino acid motif, KXFF(K/R)R, that is present in all
steroid hormone receptors and which is nearly identical to the
KXGFFKR motif of integrin subunits. Thus, via a similar
motif, calreticulin may be able to bind to two disparate families of
proteins, one in the nucleus and one at the plasma membrane, and
therefore modulate the function of these proteins.
In the present
study, we examine more closely the role of calreticulin in integrin
function. Using Jurkat T-lymphoblastoid cells, we find that
calreticulin is associated with in
cells that have been stimulated to adhere to collagen type I by
activation with monoclonal antibodies to the
and
integrin subunits. Similar results were seen when the
cells were stimulated to adhere by treatment with phorbol 12-myristate
13-acetate (PMA). (
)The stimulation of Jurkat cell adhesion
by anti-integrin antibodies or PMA was found to be additive when the
two stimuli were applied together. Thus, extracellular and
intracellular events induced both cell adhesion to integrin ligand and
the interaction of calreticulin with
in these cells. The findings of these studies suggest that the
functional state of an integrin could alter the cellular distribution
of calreticulin and/or that the functional state of an integrin may be
partially dependent upon or affected by its interaction with
calreticulin.
Figure 1:
Effect of
anti-integrin antibodies on adhesion of Jurkat cells to type I
collagen. A, Jurkat cells prelabeled with
[H]thymidine were exposed to various
anti-integrin antibodies (see Table 1) for 30 min at 37 °C
and then allowed to attach to type I collagen-coated plates. Cell
attachment was quantified as described under ``Experimental
Procedures.'' These data, obtained from quadruplicate samples, are
representative of three different experiments. Error bars in
all figures represent standard deviations about the means. B,
as in A, except prior to plating on collagen I, some cell
samples were mixed with function-blocking antibodies; either
anti-
(P1E6) or anti-
(PIB5) at 10 µg/ml final
concentration.
Figure 2:
Inducible coimmunoprecipitation of
calreticulin with integrin. A, the
integrin was
immunoprecipitated, using a Sepharose-conjugated anti-
antibody (P1E6), from the lysates of Jurkat cells which
had been preexposed to various antibodies as indicated. The
immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis, transferred to poly(vinylidene fluoride) membranes,
and analyzed by Western blot analysis as described under
``Experimental Procedures.'' The upper part of the membrane (upper panel) was probed with an anti-
antibody (P4C10), while the bottom part of the same membrane was
probed with an anti-calreticulin antibody (LAR090). The positions of
and calreticulin are indicated. Purified calreticulin
was obtained by affinity chromatography as described
previously(12) . B, the intensities of the bands in Panel A were densitometrically quantitated and the intensity
of calreticulin staining was normalized to
loading.
Figure 3:
Stimulation of attachment of Jurkat cells
to type I collagen by PMA. A,
[H]Thymidine-labeled Jurkat cells were exposed to
the indicated concentrations of PMA, and then cell attachment to type I
collagen was analyzed as described under ``Experimental
Procedures.'' B, as in A, except prior to
plating on collagen type I, some cell samples were mixed with
neutralizing anti-
antibody (PIE6) at 10
µg/ml final concentration.
In direct correlation with the PMA-induced
adhesion, Fig. 4A shows that similar treatment of
Jurkat cells resulted in increased interaction of calreticulin with
integrin . Briefly, Jurkat cells were
treated with PMA, washed, and lysed. The
integrin was immunoprecipitated from the lysates, and the
immunoprecipitates were analyzed for calreticulin by Western blotting
as in above experiments. As seen in Fig. 4A, there is a
dose-dependent increase in the amount of calreticulin associated with
in PMA-treated cells. As described
under ``Experimental Procedures,'' densitometric analyses
were performed to confirm, quantitatively, the increase in
-bound calreticulin seen in Fig. 4A. The intensity of calreticulin staining was
normalized to
and demonstrated graphically in Fig. 4B.
Figure 4:
PMA induces enhanced binding of
calreticulin to . A,
PMA-treated Jurkat cell lysates were subjected to immunoprecipitation
with an anti-
antibody as described in Fig. 2.
The immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis and Western blot analysis to detect
and calreticulin as described for Fig. 2. B, the
intensities of the bands in Panel A were densitometrically
quantitated and corrected to indicate the amount of calreticulin
relative to the amount of purified
.
Thus, PMA can induce Jurkat cells to adhere
to collagen type I via their integrin and can also trigger an interaction between this
integrin and calreticulin. These results, taken together with those
above, suggest that, when it is stimulated into adhesive activity on
Jurkat cells, the integrin
interacts
with the intracellular protein calreticulin. The results also indicate
that the concomitant induction of
function and the association of this integrin with calreticulin
can be caused by both external stimuli (e.g. activating
antibody) and internal stimuli (through PMA).
Figure 5:
Activating anti-integrin antibodies and
PMA induce a synergistic stimulation of cell attachment to collagen or
a further increase in calreticulin binding to
. A, antibody- and
PMA-induced cell attachment; B, Western blot analysis; and C, densitometric quantitation of
-associated calreticulin, were
carried out as described under ``Experimental
Procedures.''
Figure 6:
Introduction of an anti-calreticulin
antibody into Jurkat cells inhibits the ability of activating
anti-integrin antibodies and PMA to induce cell attachment to collagen.
Anti-calreticulin antibody (LAR090), anti-protein disulfide isomerase
or rabbit IgG were introduced into streptolysin O-permeablized Jurkat
cells as described previously(21) . After streptolysin O
treatment, incubation with antibodies, extensive washing and recovery,
the cells were exposed to activating anti- antibodies
or to PMA as described under ``Experimental Procedures,'' and
cell attachment to type I collagen was assayed. Solid bar,
cells pretreated with rabbit IgG; hatched bar, cells
pretreated with anti-protein disulfide isomerase antibody; clear
bar, cells pretreated with anti-calreticulin
antibody.
The results of these experiments are interesting in light of the fact that the anti-calreticulin antibody used here (LAR090) has been shown to be function-blocking. This was demonstrated by its neutralization of the interaction between calreticulin and the KXFF(K/R)R segment of steroid hormone receptors(19) . Therefore, inhibition of calreticulin binding to integrin inhibited the ability of both the activating anti-integrin antibodies and PMA to induce Jurkat cell adhesion to collagen I.
The integrins are cell surface receptors for extracellular
molecules which can transduce signals into cells as a result of binding
to their ligands. Reciprocally, cytoskeletal and biochemical events
within the cell can modify integrin function. We report here of an
interaction, between the integrin and the intracellular protein calreticulin, which may be involved
in integrin-mediated signaling and/or in the intracellular modulation
of integrin function. A functional interaction between integrins and
calreticulin has been examined previously and is known to occur at the
KXGFFKR segment of the integrin
subunit(12, 13) . In the present study, we provide
evidence that the interaction between calreticulin and integrin
is dependent upon the activation
state of the integrin and can thus be induced by reagents that activate
function. We have demonstrated that
treatment of a T-lymphoblastoid cell line, Jurkat, with activating
antibodies against the
and
integrin
subunits resulted in an increased association, above basal levels,
between
and calreticulin that was
concomitant with an increase in adhesion of these cells to type I
collagen. Similar responses were seen when the cells were treated with
PMA. In the PMA experiments, it was observed that 1 nM PMA had
a negligible effect on cell adhesion but a significant effect on
-calreticulin interaction. This is
not surprising, considering the pleiotropic effects of PMA, and may
reflect other influences that PMA has on the cells. It is also possible
that stimulation of adhesion requires at least a minimum amount of
interaction between calreticulin and
. It is feasible that, in the case of
1 nM PMA, some
-calreticulin
association is induced but that this does not meet threshold
requirements for cellular adhesion. It is important to note that Jurkat
cells do have a minimal capacity to adhere to collagen I spontaneously.
However, significant adhesion to this substrate has been shown to be
inducible through treatments with anti-
antibodies,
anti-
antibodies (JBS2 in particular) and
PMA(20) . In all cases, including the present study, induced
Jurkat adhesion to collagen type I was specifically
-mediated(20) . Furthermore,
the present study shows that both the adhesion to type I collagen and
the interaction between
and
calreticulin that were induced by treatment of Jurkat cells with a
combination of activating antibodies and PMA together were increased
above those that were seen with either treatment alone.
The results
indicate that an interaction between calreticulin and integrins, in
this case , can occur within cells
and be increased in response to specific stimuli. Moreover, the
findings we present here suggest that the interaction between
calreticulin and
is dependent upon
the activation state of the integrin and can be stimulated by both
extracellular and intracellular events. The binding of calreticulin to
is not only enhanced upon integrin
activation, but appears to be a requirement for the maintenance of the
activated state. Thus, occupation of the KXGFFKR motif by
calreticulin may stabilize the active conformation of an integrin,
since neutralization of this interaction can result in the inhibition
of integrin-mediated adhesion.
Preliminary antibody-binding studies
with Jurkat cells indicate that JBI and 6F4 competitively inhibit the
adherence of one another to cell surface suggesting
that the target epitopes are in close proximity. In contrast, B3B11 and
JBS2 each recognize distinct epitopes from each of the other
antibodies. (
)Collectively these results suggest that there
are multiple regions of the integrin which are involved in the control
of molecular activity. The epitope recognized by B3B11 and competing
antibodies has been localized to a region within approximately 75 amino
acids of the membrane adjacent to a predicted long range disulfide bond
between the C7 and C456.
While the exact location of the
epitope recognized by AIIB2 binding is not known, competitive binding
studies indicate that these two antibodies recognize distinct epitopes. (
)Thus the inhibitory effects of AIIB2 on B3B11 induced
association of calreticulin with
would seem to
suggest that the antibody is either interfering with
activation or delivering a negative signal(23) . It is
interesting to note that AIIB2, while inhibiting Jurkat adherence to
collagen and fibronectin, induces homotypic cellular aggregation
implying that interaction with this antibody may be inducing an
alternate pattern of cellular response.
In participating in
signaling pathways, the direct protein-protein interactions in which
integrins partake within the cell remain relatively elusive. Using
mainly in vitro biochemical methods, the integrin subunit cytoplasmic domain has been shown to interact
with the cytoskeletal proteins talin (10) and
-actinin(9) . Intracellular interactions involving
integrin
subunits are less well studied, however, and the
interaction between integrin
and
calreticulin described here may be the first demonstration of an
interaction between the cytoplasmic domain of an integrin
subunit
and an intracellular protein that results in the transmission of
signals into cells. While these results are largely correlative, it has
recently been shown by immunofluorescent staining that calreticulin and
, which is also a collagen receptor,
can be induced to colocalize into distinct compartments(13) .
It is also known that antisense down-regulation of calreticulin
expression leads to a reduction in cell adhesion in P19 embryonal
carcinoma cells and PC-3 prostatic carcinoma cells(13) .
Furthermore, we have previously shown that antisense down-regulation of
calreticulin abrogated the ability of adhesion-stimulating antibodies
to induce adhesion of Jurkat cells in experiments similar to those
described here (13) . Such findings are corroborated by the
data, presented here in Fig. 6, showing that intracellular
disruption of calreticulin function in Jurkat cells interfered with the
ability of these cells to be induced to adhere to collagen type I.
Thus, it is possible that calreticulin is, at the least, a transitory
component in the establishment of fully functional adhesive apparatuses
at the inner cell membrane. In light of recent studies showing a
specific function for calreticulin in regulating the activity of
steroid hormone receptors(18, 19) , it is also
plausible that the sequestering of a small intracellular pool of
calreticulin by active integrins could have an effect on the expression
of steroid hormone responsive genes and thus have a significant impact
on cell growth and differentiation. Alternatively, the interaction
between calreticulin and integrin
may influence other integrin-protein interactions or possibly
mediate biochemical events such as calcium fluxes.
Because studies
showing interactions involving the cytoplasmic domains of integrin
subunits are less well developed than those for the
subunit,
subunit cytoplasmic domains have often been
considered to be primarily regulatory in their function. One model has
the
subunit cytoplasmic domain maintaining the integrin in a low
affinity state through the KLGFFKR ``hinge''
region(11) , an example of inside-out signaling. Experimental
modification of this regulatory function, by removing the GFFKR
segment, can result in an activated, high affinity ligand-binding
integrin receptor(11) . It is reasonable to speculate that
there may be physiological interactions between integrin
subunit
cytoplasmic domains and intracellular proteins that can cause similar
``inside-out'' modifications of integrin function through
undetermined mechanisms. In support of this there have been several
reports of intracellular events which modulate the functional activity
of integrin receptors, including phorbol ester stimulation of CHO cells (25) and stimulation of platelets with
agonists(24, 26) . However, like the cytoplasmic
associations that integrins form as a result of ligand binding, the
exact molecular mechanisms by which intracellular events modify
integrin function remain mostly unclear. Based on the results presented
here, it is possible that calreticulin plays a physiological role in
moderating integrin function. As such, calreticulin could be seen as
binding to the integrin
subunit, via KXGFFKR, after
integrin activation and helping to stabilize the integrin in an
activated conformation.
As well as a novel mechanism for the
regulation of integrin function the data here suggest a novel function
for calreticulin. While primarily characterized as a major
calcium-binding protein in the lumen of the endoplasmic
reticulum(17) , recent observations suggest that calreticulin
has many functions. In addition to the steroid hormone receptor-binding
activity described above, calreticulin has been shown to form a tight
complex with flavin-containing mono-oxygenase (27) and has also
been shown to have RNA binding activity(28) . Some, but not
all, of these functions for calreticulin involve calreticulin that is
not necessarily associated with endoplasmic reticulum membranes.
Localization of calreticulin to sites within cells other than the
endoplasmic reticulum has been reported
previously(13, 16, 17) , and it is becoming
clear that, despite containing a KDEL endoplasmic reticulum retention
sequence, the intracellular distribution of calreticulin is not so
strictly limited. Other KDEL containing proteins have also been shown
to have nonendoplasmic reticulum localizations. These include protein
disulfide-isomerase(29) , endoplasmin (30) and the
heavy chain binding protein (BiP/GRP78)(30) . We have confirmed
that the calreticulin which we found associated with
in the present study is recognized
by an anti-KDEL antiserum. (
)
The biochemical mechanisms
which govern the cellular distribution of calreticulin as well as those
which control the inducible interaction with integrins still remain to
be elucidated. It is possible that post-translational modifications to
calreticulin are important in determining these cellular activities.
With regard to its RNA-binding capacity it has been shown that
phosphorylation of calreticulin is necessary for its binding of
RNA(28) . We have not determined if the calreticulin that is
associated with integrins is phosphorylated, but appropriate studies
are under way. Such investigations are interesting in light of the
findings presented here involving PMA. Calreticulin does contain a
putative site for phosphorylation by protein kinase C (17) and
it remains to be determined if this site is utilized in vivo.
It is possible that activation of protein kinase C results in the
stimulation of intracellular signaling pathways involving calreticulin,
its phosphorylation and its interaction with other proteins. Some of
the associated activities of calreticulin may, in turn, be intimately
linked to cell adhesion. We have presented here evidence which suggests
that calreticulin, through its interaction with the integrin
, may play a role in the adhesive
activities of this integrin. The calreticulin-integrin interaction may
facilitate integrin function and/or this interaction may affect
downstream consequences of integrin ligation. Presently, investigation
of the calreticulin-integrin interaction is ongoing to determine the
exact function this association has within cells.