Limits...
Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling.

Xi X, Bodnar RJ, Li Z, Lam SC, Du X - J. Cell Biol. (2003)

Bottom Line: A point mutation replacing Y759 with alanine also abolished integrin activation.Thus, the T755NITY759 sequence of beta3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling.In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of beta3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.

ABSTRACT
Bidirectional signaling of integrin alphaIIbbeta3 requires the beta3 cytoplasmic domain. To determine the sequence in the beta3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin alphaIIb, and mutants of beta3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of beta3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of beta3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of beta3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.

Show MeSH

Related in: MedlinePlus

Progressive cleavage at different sites of the β3 cytoplasmic domain by calpain. Washed human platelets (109/ml) were directly solubilized in SDS-PAGE sample buffer containing EDTA and E64 (Control), or treated with thrombin (0.1 U/ml) for increasing lengths of time at 37°C before being solubilized. Platelet lysates were then immunoblotted with Ab759, Ab754, Ab747, and Ab741 to detect calpain cleavages at Y759, F754, T747, and Y741 sites, with Ab762 to detect uncleaved β3 and Mab 15 to detect total β3 levels. Quantification of antibody reactions was performed by scanning the immunoblots and analyzing using NIH Image as described under the Materials and methods. Percentages of the molecules cleaved (or uncleaved) at a particular site are shown in B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172800&req=5

fig7: Progressive cleavage at different sites of the β3 cytoplasmic domain by calpain. Washed human platelets (109/ml) were directly solubilized in SDS-PAGE sample buffer containing EDTA and E64 (Control), or treated with thrombin (0.1 U/ml) for increasing lengths of time at 37°C before being solubilized. Platelet lysates were then immunoblotted with Ab759, Ab754, Ab747, and Ab741 to detect calpain cleavages at Y759, F754, T747, and Y741 sites, with Ab762 to detect uncleaved β3 and Mab 15 to detect total β3 levels. Quantification of antibody reactions was performed by scanning the immunoblots and analyzing using NIH Image as described under the Materials and methods. Percentages of the molecules cleaved (or uncleaved) at a particular site are shown in B.

Mentions: We have shown previously that calpain may cleave the cytoplasmic domain of β3 at sites COOH terminal to T741, Y747, F754, and Y759, which generates β3 fragments identical to the above-described truncation mutants of β3 (741, 747, 754, and 759). As we showed above that truncations at these different sites result in different functional effects on integrin signaling, our results also indicate that cleavage of the β3 cytoplasmic domain at these different sites has the potential to differentially regulate outside-in and inside-out signaling of integrin αIIbβ3. To determine if calpain differentially cleaves the β3 integrin at different sites during platelet activation, washed platelets were treated with or without thrombin (0.1 U/ml) and then immunoblotted for calpain cleavage by the cleavage-specific antibodies (Du et al., 1995). We found that anti-759 antibody, which only recognizes β3 molecules with cleavage at Y759, reacted with ∼0.8% of the integrin αIIbβ3 molecules in washed “resting” platelets (Fig. 7). This reaction was unlikely to result from cross-reaction of this antibody with the intact β3 subunit, because we showed that the antibody did not react with the intact β3 subunit but reacted with the Δ759 mutant expressed in CHO cells (Fig. 2). Thus a very small percentage of the β3 molecules in resting platelets has been cleaved at the Y759 site. Stimulation of platelets with thrombin caused a time-dependent and significant increase in the cleavage at Y759. In contrast to the 759 site, calpain cleavage at T754 or Y747 (Fig. 7) occurred to a much lesser degree and only after a much longer exposure to thrombin. Calpain cleavage at the 741 site was not detectable in thrombin-stimulated platelets (unpublished data) but was detected in platelets treated with calcium ionophore A23187 (Du et al., 1995). Thus, in thrombin-activated platelets, calpain preferentially cleaves β3 at Y759. As we showed above that cleavage at Y759 selectively reduced integrin outside-in signaling without affecting inside-out signaling, this result indicates that calpain cleavage has the potential to selectively regulate outside-in signaling during platelet activation.


Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling.

Xi X, Bodnar RJ, Li Z, Lam SC, Du X - J. Cell Biol. (2003)

Progressive cleavage at different sites of the β3 cytoplasmic domain by calpain. Washed human platelets (109/ml) were directly solubilized in SDS-PAGE sample buffer containing EDTA and E64 (Control), or treated with thrombin (0.1 U/ml) for increasing lengths of time at 37°C before being solubilized. Platelet lysates were then immunoblotted with Ab759, Ab754, Ab747, and Ab741 to detect calpain cleavages at Y759, F754, T747, and Y741 sites, with Ab762 to detect uncleaved β3 and Mab 15 to detect total β3 levels. Quantification of antibody reactions was performed by scanning the immunoblots and analyzing using NIH Image as described under the Materials and methods. Percentages of the molecules cleaved (or uncleaved) at a particular site are shown in B.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172800&req=5

fig7: Progressive cleavage at different sites of the β3 cytoplasmic domain by calpain. Washed human platelets (109/ml) were directly solubilized in SDS-PAGE sample buffer containing EDTA and E64 (Control), or treated with thrombin (0.1 U/ml) for increasing lengths of time at 37°C before being solubilized. Platelet lysates were then immunoblotted with Ab759, Ab754, Ab747, and Ab741 to detect calpain cleavages at Y759, F754, T747, and Y741 sites, with Ab762 to detect uncleaved β3 and Mab 15 to detect total β3 levels. Quantification of antibody reactions was performed by scanning the immunoblots and analyzing using NIH Image as described under the Materials and methods. Percentages of the molecules cleaved (or uncleaved) at a particular site are shown in B.
Mentions: We have shown previously that calpain may cleave the cytoplasmic domain of β3 at sites COOH terminal to T741, Y747, F754, and Y759, which generates β3 fragments identical to the above-described truncation mutants of β3 (741, 747, 754, and 759). As we showed above that truncations at these different sites result in different functional effects on integrin signaling, our results also indicate that cleavage of the β3 cytoplasmic domain at these different sites has the potential to differentially regulate outside-in and inside-out signaling of integrin αIIbβ3. To determine if calpain differentially cleaves the β3 integrin at different sites during platelet activation, washed platelets were treated with or without thrombin (0.1 U/ml) and then immunoblotted for calpain cleavage by the cleavage-specific antibodies (Du et al., 1995). We found that anti-759 antibody, which only recognizes β3 molecules with cleavage at Y759, reacted with ∼0.8% of the integrin αIIbβ3 molecules in washed “resting” platelets (Fig. 7). This reaction was unlikely to result from cross-reaction of this antibody with the intact β3 subunit, because we showed that the antibody did not react with the intact β3 subunit but reacted with the Δ759 mutant expressed in CHO cells (Fig. 2). Thus a very small percentage of the β3 molecules in resting platelets has been cleaved at the Y759 site. Stimulation of platelets with thrombin caused a time-dependent and significant increase in the cleavage at Y759. In contrast to the 759 site, calpain cleavage at T754 or Y747 (Fig. 7) occurred to a much lesser degree and only after a much longer exposure to thrombin. Calpain cleavage at the 741 site was not detectable in thrombin-stimulated platelets (unpublished data) but was detected in platelets treated with calcium ionophore A23187 (Du et al., 1995). Thus, in thrombin-activated platelets, calpain preferentially cleaves β3 at Y759. As we showed above that cleavage at Y759 selectively reduced integrin outside-in signaling without affecting inside-out signaling, this result indicates that calpain cleavage has the potential to selectively regulate outside-in signaling during platelet activation.

Bottom Line: A point mutation replacing Y759 with alanine also abolished integrin activation.Thus, the T755NITY759 sequence of beta3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling.In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of beta3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.

ABSTRACT
Bidirectional signaling of integrin alphaIIbbeta3 requires the beta3 cytoplasmic domain. To determine the sequence in the beta3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin alphaIIb, and mutants of beta3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of beta3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of beta3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of beta3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.

Show MeSH
Related in: MedlinePlus