©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inducible Interaction of Integrin with Calreticulin
DEPENDENCE ON THE ACTIVATION STATE OF THE INTEGRIN (*)

(Received for publication, May 15, 1995; and in revised form, June 21, 1995)

Marc Coppolino (1) Chungyee Leung-Hagesteijn (1) Shoukat Dedhar (1)(§) John Wilkins (2)

From the  (1)Division of Cancer Biology Research, Sunnybrook Health Science Centre, Toronto, Ontario, M4N 3M5, Canada and (2)RDU Research Laboratory Medicine, Winnipeg, Manitoba K3A 1M4, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously demonstrated an interaction between the highly conserved KXGFFKR sequence of the integrin alpha-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 alpha(2)beta(1) 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. alpha(2)beta(1) activation with either anti-beta(1) or anti-alpha(2) 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 alpha(2)beta(1) 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 alpha(2)beta(1) or by PMA. These data suggest that the active, high affinity form of alpha(2)beta(1) binds calreticulin and that calreticulin binding to the alpha(2) 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 alpha subunit of the integrin.


INTRODUCTION

The integrins are a family of heterodimeric, transmembrane glycoproteins that are formed by the noncovalent association of alpha and beta 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 alpha and beta 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 beta(1) subunit. The cytoplasmic domain of the beta(1) subunit has been shown to interact with components of the actin-based cytoskeleton, namely alpha-actinin (9) and talin(10) . In contrast to these beta subunit-specific functions, cytoplasmic interactions involving the alpha subunits have not been as well characterized.

The cytoplasmic domains of the integrin alpha subunits are diverse in both length and amino acid composition. The only generally conserved feature within the alpha subunit cytoplasmic domains is the KXGFFKR motif which immediately follows the membrane spanning domain(3, 8) . The conservation of this motif in all alpha 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 alpha subunit-cytoplasmic protein association, the KXGFFKR motif of integrin alpha 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 alpha 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 alpha(2)beta(1) in cells that have been stimulated to adhere to collagen type I by activation with monoclonal antibodies to the alpha(2) and beta(1) integrin subunits. Similar results were seen when the cells were stimulated to adhere by treatment with phorbol 12-myristate 13-acetate (PMA). (^1)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 alpha(2)beta(1) 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.


EXPERIMENTAL PROCEDURES

Materials

[^3H]Thymidine was purchased from DuPont NEN. The anti-alpha(2) integrin (P1E6), anti-alpha(3) integrin (P1B5), and anti-beta(1) integrin (P4C10) monoclonal antibodies were purchased from Life Technologies, Inc. The JB1, 6F4, and B3B11 anti-beta(1) as well as the JBS2 anti-alpha(2) monoclonal antibodies were prepared as described elsewhere(20) . The anti-beta(1) integrin monoclonal antibody AIIB2 was provided by Dr. Carolyn Damsky (University of California, San Francisco, CA). The anti-calreticulin polyclonal antiserum LAR090 was obtained from Dr. Luis A. Rokeach (University of Montreal, Montreal, PQ). Anti-protein disulfide isomerase antibody was a kind gift from Dr. Marek Michalak (University of Alberta, Edmonton, AL). Collagen type I was purchased from Sigma and CNBr-activated Sepharose from Pharmacia Biotech Inc. Secondary antibodies were purchased from Jackson Immunoresearch Laboratories. The properties of the (noncommercial) anti-integrin antibodies used here are described in Table 1.



Cell Culture and Activation

Human Jurkat cells (clone E6.3) were grown in suspension culture in RPMI 1640 containing 5% fetal calf serum and 5 times 10 M 2-mercaptoethanol. Prior to analysis, cells were harvested from culture by centrifugation, washed in serum-free RPMI 1640, and resuspended in serum-free RPMI 1640. The cells were then incubated at 37 °C with occasional mixing for 30 min in the presence of various antibodies (10 µg/ml final) or PMA (1-10 nM). Following this incubation period, the cells were washed twice in phosphate-buffered saline (100 mM NaCl, 10 mM sodium phosphate, pH 7.3) and then analyzed for cell attachment or lysed for use in protein analyses.

Cell Attachment Assays

The collagen type I was suspended at 10 µg/ml in phosphate-buffered saline and 100 µl/well were used to precoat 96-well plates (Linbro/Titertek, McLean, VA) at 4 °C overnight. Wells were washed and then blocked at 37 °C for 2 h with wash buffer (RPMI 1640, 2.5 mg/ml bovine serum albumin). Jurkat cells were prelabeled with [^3H]thymidine (1 µCi/ml of culture medium) at 37 °C overnight. After activation, 5 times 10^4 cells were seeded into quadruplicate wells and incubated at 37 °C for 3 h. Plates were then gently inverted to remove nonadherent cells and washed twice with 150 µl of wash buffer. The remaining adherent cells were lysed and attachment was quantitated by scintillation counting of ^3H.

Protein Analysis

Antibody- and PMA-treated Jurkat cells were lysed in radioimmuno precipitation buffer (phosphate-buffered saline, pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Lysates were cleared by centrifugation and protein concentrations were determined with the Bio-Rad protein assay. Equivalent amounts of protein were immunoprecipitated with anti-alpha(2) antibody (P1E6) that had been conjugated to CNBr-activated Sepharose beads. Immunoprecipitated protein complexes were washed twice with radioimmuno precipitation buffer containing 0.5 M NaCl and then twice with radioimmuno precipitation buffer alone. The immunoprecipitated proteins were resuspended by boiling in Laemmli sample buffer and then separated by SDS-polyacrylamide (7.5%) gel electrophoresis under nonreducing conditions. Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore, Bedford, MA), and the membranes were then blocked with 4% bovine serum albumin in TBS-T (20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween 20, pH 7.6). The membranes were cut in two, and the top portions were probed with the mouse anti-human beta(1) monoclonal antibody P4C10 while the bottom portions were probed with the rabbit anti-human calreticulin antiserum LAR090. Species-specific horseradish peroxidase-conjugated secondary antibodies were then applied, and detection was performed using the enhanced chemiluminescence (ECL) detection system (Amersham Corp.) following the manufacturer's instructions. The relative intensities of protein bands in the Western blots were determined using an LKB laser densitometer (model 2222-020) and Gelscan XL software (Pharmacia).

Cell Permeabilization and Antibody Internalization

[^3H]Thymidine-labeled Jurkat cells were washed and exposed to streptolysin O (100 µg/ml) at 37 °C for 30 min(21) . The cells were then washed four times with streptolysin O buffer (21) (137 mM NaCl, 2.7 mM KCl, 20 mM PIPES, 5.6 mM glucose, pH 7.0) and resuspended with either rabbit IgG (50 µg/ml) or protein A-purified anti-calreticulin antibody LAR090 (50 µg/ml). After incubation for 1 h at 37 °C, the cells were washed extensively with serum-free medium, resuspended, and activated with the anti-integrin antibodies as described above.


RESULTS

Jurkat Cell Adhesion to Type I Collagen Can Be Stimulated by Treatment with Epitope-specific Anti-integrin Antibodies

Jurkat cells express the integrin alpha(2)beta(1) on their surface, but display only minimal spontaneous adherence to collagen type I(22) . Previous investigations have shown that treatment of Jurkat cells with anti-integrin antibodies can induce these cells to adhere to collagen type I in an integrin alpha(2)beta(1)-dependent manner(20, 23) . Recently, we have characterized several monoclonal antibodies (JB1, B3B11, 6F4, JBS2) which recognize specific epitopes on the alpha(2) or the beta(1) integrin subunits (24) . (^2)In the present study we show that these antibodies have differing capacities to induce Jurkat cells to adhere to collagen type I. Fig. 1A shows that treatment of Jurkat cells with JB1, 6F4, B3B11, JBS2, or combinations of these antibodies resulted in the stimulation of adhesion of these cells to type I collagen. When the cells were concomitantly treated with the inhibitory anti-beta(1) antibody AIIB2, this induced adhesion to collagen type I was reduced. As shown previously(20) , and again here in Fig. 1B, the adhesion induced by stimulation of Jurkat cells with both anti-alpha(2) and anti-beta(1)antibodies is completely alpha(2) dependent. Fig. 1B represent experiments wherein Jurkat cells were stimulated to adhere to collagen I with either anti-alpha(2) antibody (JBS2) or anti-beta(1) antibodies (JB1 and B3B11). In each case, the induced adhesion could be neutralized by the presence of the inhibitory anti-alpha(2) antibody PIE6. In contrast to this, a function-blocking anti-alpha(3) antibody, P1B5, had no effect on the inducible adhesion of Jurkat cells to collagen type I. These data demonstrate that the binding of certain antibodies to the alpha(2)beta(1) integrin on Jurkat cells can induce the adhesion of these cells to type I collagen, possibly by causing conformational changes in this integrin. This induced adhesion can be abrogated by the presence of the function-blocking antibodies AIIB2 or P1E6.


Figure 1: Effect of anti-integrin antibodies on adhesion of Jurkat cells to type I collagen. A, Jurkat cells prelabeled with [^3H]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-alpha(2) (P1E6) or anti-alpha(3) (PIB5) at 10 µg/ml final concentration.



Treatment of Jurkat Cells with Epitope-Specific Anti-integrin Antibodies Results in the Interaction of Calreticulin with Integrin alpha(2)beta(1)

Calreticulin has been shown to interact with the KLGFFKR segment of integrins and with intact, labeled, integrin subunits in vitro(12, 13) . Calreticulin has also been shown to colocalize with clustered integrins at the plasma membrane(13) . Therefore, we decided to examine the relationship between calreticulin and the integrin alpha(2)beta(1) in Jurkat cells that had been induced to adhere to collagen I with specific antibodies. As in the adhesion experiments described in Fig. 1, Jurkat cells were treated with epitope-specific antibodies raised against either the alpha(2) or the beta(1) integrin subunit. The cells were then lysed and the alpha(2)beta(1) integrin was immunoprecipitated from the lysates. Immunoprecipitated proteins were then analyzed for the presence of calreticulin by Western blotting as described under ``Experimental Procedures.'' Fig. 2A shows that an increased amount of calreticulin was found in association with alpha(2)beta(1) integrin in cells that were treated with conditions that induced adhesion to collagen I. In contrast, little calreticulin is seen in the anti-alpha(2) immunoprecipitates of Jurkat cells that were not stimulated to adhere to collagen type I. In Fig. 2B, densitometric analysis of the Western blot in Fig. 2A is shown. The amount of calreticulin was quantified as described under ``Experimental Procedures,'' normalized to the amount of integrin that had been immunoprecipitated and displayed graphically. Thus, in these cells, increased calreticulin was found in association with integrin alpha(2)beta(1) that had been stimulated into adhesive activity with specific antibodies, while basal levels of calreticulin were found in association with alpha(2)beta(1) on nonadherent cells. Furthermore, the function-blocking antibody AIIB2 not only inhibited adhesion of Jurkat cells, but also decreased to basal levels the amount of alpha(2)beta(1)-bound calreticulin that could be induced with activating antibodies.


Figure 2: Inducible coimmunoprecipitation of calreticulin with alpha(2)beta(1) integrin. A, the alpha(2)beta(1) integrin was immunoprecipitated, using a Sepharose-conjugated anti-alpha(2) 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-beta(1) antibody (P4C10), while the bottom part of the same membrane was probed with an anti-calreticulin antibody (LAR090). The positions of beta(1) 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 beta(1) loading.



Treatment of Jurkat Cells with PMA Induces Both Adhesion to Type I Collagen and Interaction between Calreticulin and alpha(2)beta(1) Integrin

It has been previously shown that PMA can induce the adhesion of Jurkat cells to collagen type I and that this induced adhesion is alpha(2)beta(1)-mediated(20, 22, 23) . These and the above results led us to investigate whether there was any interaction between calreticulin and the integrin alpha(2)beta(1) on Jurkat cells that had been stimulated to adhere to type I collagen via treatment with PMA. As in experiments described previously, [^3H]thymidine-labeled Jurkat cells were treated with PMA, washed, and applied to adhesion assays on type I collagen. In agreement with previous studies(20) , Fig. 3A shows that treatment of Jurkat cells with 1 nM PMA had minimal effect on cell adhesion, whereas treatment with 10 nM PMA resulted in approximately a 300% increase in Jurkat cell adhesion to collagen I. Fig. 3B demonstrates the alpha(2)beta(1) specificity of this PMA-induced adhesion. Greater than 80% of the adhesion induced by 10 nM PMA was abrogated by the inhibitory anti-alpha(2) antibody PIE6.


Figure 3: Stimulation of attachment of Jurkat cells to type I collagen by PMA. A, [^3H]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-alpha(2) 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 alpha(2)beta(1). Briefly, Jurkat cells were treated with PMA, washed, and lysed. The alpha(2)beta(1) 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 alpha(2)beta(1) in PMA-treated cells. As described under ``Experimental Procedures,'' densitometric analyses were performed to confirm, quantitatively, the increase in alpha(2)beta(1)-bound calreticulin seen in Fig. 4A. The intensity of calreticulin staining was normalized to beta(1) and demonstrated graphically in Fig. 4B.


Figure 4: PMA induces enhanced binding of calreticulin to alpha(2)beta(1). A, PMA-treated Jurkat cell lysates were subjected to immunoprecipitation with an anti-alpha(2) antibody as described in Fig. 2. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and Western blot analysis to detect beta(1) 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 beta(1).



Thus, PMA can induce Jurkat cells to adhere to collagen type I via their alpha(2)beta(1) 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 alpha(2)beta(1) interacts with the intracellular protein calreticulin. The results also indicate that the concomitant induction of alpha(2)beta(1) 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).

The Effects of Anti-integrin Antibodies and PMA on Both Jurkat Cell Adhesion and Calreticulin-alpha(2)beta(1) Interaction Are Additive

To determine if the activated adhesion or the induced interaction between calreticulin and alpha(2)beta(1) that we observed to be caused by treatment with either anti-integrin antibodies or PMA were maximal, we treated Jurkat cells with a combination of epitope-specific anti-integrin antibodies and PMA. The Jurkat cells were treated as in previous experiments except that some samples were incubated with both anti-integrin antibodies (B3B11 and JBS2) and PMA (10 nM) prior to either plating for adhesion assays or lysis for alpha(2)beta(1) immunoprecipitation and Western blotting. Fig. 5shows that the stimulation of Jurkat cell adhesion to type I collagen (A) and the amount of calreticulin co-purified in anti-alpha(2)beta(1) immunoprecipitates (B) was greatest when the Jurkat cells were treated with a combination of B3B11, JBS2, and 10 nM PMA. Importantly, the level of adhesion seen and the amount of calreticulin copurified with alpha(2)beta(1) under these conditions were greater than when Jurkat cells were treated with either of these reagents alone. Again, densitometric quantification corrected for beta(1) loading and is shown in Fig. 5C. The results of the adhesion experiments are in agreement with previous studies(20) , and together these results suggest that both the activation of integrin function and the induced calreticulin-alpha(2)beta(1) interaction can be achieved via two different pathways, one stimulated by PMA and another mimicked by the anti-integrin antibodies. Moreover, these two pathways to integrin activation can be additive.


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 alpha(2)beta(1). A, antibody- and PMA-induced cell attachment; B, Western blot analysis; and C, densitometric quantitation of alpha(2)beta(1)-associated calreticulin, were carried out as described under ``Experimental Procedures.''



Introduction of Anti-calreticulin Antibodies into Jurkat Cells Results in Partial Inhibition of the Adhesion to Type I Collagen Induced by Antibodies or PMA

In order to determine whether the binding of calreticulin to alpha(2) was required for integrin activation, we introduced a purified anti-calreticulin antibody (LAR090) into streptolysin O-permeablized Jurkat cells prior to the integrin activation. Streptolysin O did not have any effect on cell viability as has been previously demonstrated by others(21) . As shown in Fig. 6, neither activating anti-beta(1) antibodies nor PMA (10 nM) could stimulate adhesion to type I collagen in Jurkat cells which had been pretreated with the anti-calreticulin antibody. Both purified rabbit IgG and purified anti-protein disulfide isomerase had no effect on anti-beta(1)- or PMA-induced adhesion when introduced into streptolysin O-treated cells. Like calreticulin, protein disulfide isomerase is an intracellular protein which contains a KDEL sequence and was thus targeted as an appropriate negative control.


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-beta(1) 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.


DISCUSSION

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 alpha(2)beta(1) 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 alpha subunit(12, 13) . In the present study, we provide evidence that the interaction between calreticulin and integrin alpha(2)beta(1) is dependent upon the activation state of the integrin and can thus be induced by reagents that activate alpha(2)beta(1) function. We have demonstrated that treatment of a T-lymphoblastoid cell line, Jurkat, with activating antibodies against the alpha(2) and beta(1) integrin subunits resulted in an increased association, above basal levels, between alpha(2)beta(1) 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 alpha(2)beta(1)-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 alpha(2)beta(1). It is feasible that, in the case of 1 nM PMA, some alpha(2)beta(1)-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-beta(1) antibodies, anti-alpha(2) antibodies (JBS2 in particular) and PMA(20) . In all cases, including the present study, induced Jurkat adhesion to collagen type I was specifically alpha(2)beta(1)-mediated(20) . Furthermore, the present study shows that both the adhesion to type I collagen and the interaction between alpha(2)beta(1) 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 alpha(2)beta(1), 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 alpha(2)beta(1) is dependent upon the activation state of the integrin and can be stimulated by both extracellular and intracellular events. The binding of calreticulin to alpha(2)beta(1) 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 beta(1) suggesting that the target epitopes are in close proximity. In contrast, B3B11 and JBS2 each recognize distinct epitopes from each of the other antibodies. (^3)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.^2 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. (^4)Thus the inhibitory effects of AIIB2 on B3B11 induced association of calreticulin with alpha(2) would seem to suggest that the antibody is either interfering with beta(1) 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 beta(1) integrin subunit cytoplasmic domain has been shown to interact with the cytoskeletal proteins talin (10) and alpha-actinin(9) . Intracellular interactions involving integrin alpha subunits are less well studied, however, and the interaction between integrin alpha(2)beta(1) and calreticulin described here may be the first demonstration of an interaction between the cytoplasmic domain of an integrin alpha 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 alpha(3)beta(1), 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 alpha(2)beta(1) 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 alpha subunits are less well developed than those for the beta(1) subunit, alpha subunit cytoplasmic domains have often been considered to be primarily regulatory in their function. One model has the alpha 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 alpha 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 alpha 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 alpha(2)beta(1) in the present study is recognized by an anti-KDEL antiserum. (^5)

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 alpha(2)beta(1), 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Cancer Biology Research, Reichmann Research Bldg., S-218 Sunnybrook Health Science Centre, 2075 Bayview Ave., Toronto, Ontario, Canada M4N 3M5. Tel.: 416-480-5711; Fax: 416-480-5703.

(^1)
The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
J. Wilkins, D. Stupack, H. Ni, N. Hunt and C. Shen, manuscript submitted for publication.

(^3)
J. Wilkins and D. Stupack, unpublished data.

(^4)
J. Wilkins and D. Stupack, unpublished observations.

(^5)
M. Coppolino and S. Dedhar, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Luis Rokeach (University of Montreal) for his kind gift of anti-calreticulin antibodies and Lynda Woodcock and Terrie Walker for preparing the manuscript.


REFERENCES

  1. Hynes, R. O. (1992) Cell 69,11-25 [Medline] [Order article via Infotrieve]
  2. Ruoslahti, E., and Pierschbacher, M. D. (1987) Science 238,491-497 [Medline] [Order article via Infotrieve]
  3. Dedhar, S. (1990) BioEssays 12,583-590 [Medline] [Order article via Infotrieve]
  4. Juliano, R. L., and Haskill, S. (1993) J. Cell Biol. 120,577-585 [Medline] [Order article via Infotrieve]
  5. Springer, T. A. (1990) Nature 346,425-434 [CrossRef][Medline] [Order article via Infotrieve]
  6. Symington, B. E., Takada, Y., and Carter, W. G. (1993) J. Cell Biol. 120,523-535 [Abstract]
  7. Dedhar, S. (1994) Trends Biochem. Sci. 19,269-271 [CrossRef][Medline] [Order article via Infotrieve]
  8. Williams, M. J., Hughes, P. E., O'Toole, T. E., and Ginsberg, M. H. (1994) Trends Cell Biol. 4,109-112 [CrossRef]
  9. Otey, C. A., Pavalko, F. M., and Burridge, K. (1990) J. Cell Biol. 111,721-729 [Abstract]
  10. Horwitz, A., Duggan, K., Buck, C., Beckerle, M. C., and Burridge, K. (1986) Nature 320,531-533 [Medline] [Order article via Infotrieve]
  11. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H. (1994) J. Cell Biol. 124,1047-1059 [Abstract]
  12. Rojiani, M. V., Finlay, B. B., Gray, V., and Dedhar, S. (1991) Biochemistry 30,9859-9866 [Medline] [Order article via Infotrieve]
  13. Leung-Hagesteijn, C. Y., Milankov, K., Michalak, M., Wilkins, J., and Dedhar, S. (1994) J. Cell Sci. 107,589-600 [Abstract/Free Full Text]
  14. MacLennan, D. H., and Wong, P. T. (1972) Proc. Natl. Acad. Sci. U. S. A. 68,1231-1235
  15. Ostwald, T. J., and MacLennan, D. H. (1974) J. Biol. Chem. 249,974-979 [Abstract/Free Full Text]
  16. Opas, M., Dziak, E., Fliegel, L., and Michalak, M. (1991) J. Cell. Physiol. 149,160-171 [Medline] [Order article via Infotrieve]
  17. Michalak, M., Milner, R. E., Burns, K., and Opas, M. (1992) Biochem. J. 285,681-692 [Medline] [Order article via Infotrieve]
  18. Burns, K., Duggan, B., Atkinson, E. A., Famulski, K. S., Nemer, M., Bleackley, R. C., and Michalak, M. (1994) Nature 367,476-480 [CrossRef][Medline] [Order article via Infotrieve]
  19. Dedhar, S., Rennie, P. S., Shago, M., Leung-Hagesteijn, C. Y., Yang, H., Filmus, J., Hawley, R. G., Bruchovsky, N., Cheng, H., Matusik, R. J., and Giguere, V. (1994) Nature 367,480-483 [CrossRef][Medline] [Order article via Infotrieve]
  20. Stupack, D. G., Shen, C., and Wilkins, J. A. (1994) Cell. Immunol. 155,237-245 [CrossRef][Medline] [Order article via Infotrieve]
  21. Pacifici, R., Roman, J., Kimble, R., Civitelli, R., Brownfield, C. M., and Bizzarri, C. (1994) J. Immunol. 153,2222-2233 [Abstract/Free Full Text]
  22. Wilkins, J. A., Stupack, D., Stewart, S., and Caixia, S. (1991) Eur. J. Immunol. 21,517-522 [Medline] [Order article via Infotrieve]
  23. Kapron-Bras, C., Fitz-Gibbon, L., Jeevaratnam, P., Wilkins, J., and Dedhar, S. (1993) J. Biol. Chem. 268,20701-20704 [Abstract/Free Full Text]
  24. Sims, P. J., Ginsberg, M. H., Plow, E. F., and Shattil, S. J. (1991) J. Biol. Chem. 266,7345-7352 [Abstract/Free Full Text]
  25. Danilov, Y. N., and Juliano, R. L. (1989) J. Cell Biol. 108,1925-1933 [Abstract]
  26. Shattil, S. J., and Brugge, J. S. (1991) Curr. Opin. Cell Biol. 3,869-879 [Medline] [Order article via Infotrieve]
  27. Guan, S., Falick, A. M., Williams, D. E., and Cashman, J. R. (1991) Biochemistry 30,9892-9900 [Medline] [Order article via Infotrieve]
  28. Singh, N. K., Atreya, C. D., and Nakhasi, H. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,12770-12774 [Abstract/Free Full Text]
  29. Yoshimori, T., Semba, T., Takemoto, H., Akagi, S., Yamamoto, A., and Tashiro, Y. (1990) J. Biol. Chem. 265,15984-15990 [Abstract/Free Full Text]
  30. Takemoto, H., Yoshimori, T., Yamamoto, A., Miyata, Y., Yahara, I., Inoue, K., and Tashiro, Y. (1992) Arch. Biochem. Biophys. 296,129-136 [Medline] [Order article via Infotrieve]

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