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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.

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Localization of calpain-cleaved β3 in spreading platelets. Platelets in Tyrode's buffer (1 × 108/ml) were incubated in the chamber slides precoated with 20 μg/ml fibrinogen for 2 h at 37°C. After three washes, the adherent platelets were fixed with 4% paraformaldehyde and permeabilized. The slides were incubated subsequently with Mab15, specific for integrin β3 extracellular domain (green), and with one of the cleavage site–specific antibodies (Ab759 and Ab754) or an antibody against the COOH terminus of β3 (Ab762) (red), and then with Alexa Fluor 488–conjugated anti–mouse IgG antibody and Alexa Fluor 546–conjugated anti–rabbit IgG antibodies. The slides were scanned under a confocal microscope (63× lens). Images of the green and red fluorescence overlay are shown in the left column. Green fluorescence (Mab15) from the same images is shown in the middle column. Red fluorescence (cleavage site–specific antibodies) from the same images is shown in the right column.
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fig8: Localization of calpain-cleaved β3 in spreading platelets. Platelets in Tyrode's buffer (1 × 108/ml) were incubated in the chamber slides precoated with 20 μg/ml fibrinogen for 2 h at 37°C. After three washes, the adherent platelets were fixed with 4% paraformaldehyde and permeabilized. The slides were incubated subsequently with Mab15, specific for integrin β3 extracellular domain (green), and with one of the cleavage site–specific antibodies (Ab759 and Ab754) or an antibody against the COOH terminus of β3 (Ab762) (red), and then with Alexa Fluor 488–conjugated anti–mouse IgG antibody and Alexa Fluor 546–conjugated anti–rabbit IgG antibodies. The slides were scanned under a confocal microscope (63× lens). Images of the green and red fluorescence overlay are shown in the left column. Green fluorescence (Mab15) from the same images is shown in the middle column. Red fluorescence (cleavage site–specific antibodies) from the same images is shown in the right column.

Mentions: Calpain only cleaves a percentage of β3 molecules in platelets stimulated with thrombin, suggesting that cleavage of β3 is likely to have only a localized effect on integrin function. It is known that during cell spreading on integrin ligands, integrin forms a localized signaling complex with cytoskeletal and signaling molecules that is dynamically regulated and involves calpain activity (Bialkowska et al., 2000). To determine if selective integrin cleavage occurred at specific regions during platelet spreading on integrin ligands, platelets were allowed to spread on fibrinogen for 60 min and then double stained with calpain cleavage–specific antibodies and a monoclonal antibody against an extracellular epitope of β3 that is not affected by calpain cleavage. Consistent with the above results in thrombin-stimulated platelets, calpain cleavage occurred mainly at Y759 (Fig. 8), but cleavages at F754 (Fig. 8) or the more NH2-terminal sites (unpublished data) were significantly weaker. Whereas the β3 molecules were distributed throughout the spreading platelets with particularly strong staining at the edges, calpain-cleaved β3 was not seen at the edges (or in the pseudopods) but was concentrated in the central region of the spreading platelets as clusters. As integrin engagement with immobilized ligands starts from the central region where discoid platelets initially adhere, our data suggest that cleavage of β3 by calpain occurred only to the population of the integrin that is no longer at the leading edge of spreading platelets. This selective cleavage of the centrally located integrin population (mainly at Y759) may serve to down-regulate local integrin outside-in signaling in these areas without affecting the activation state of the integrin. It is possible that selective cleavage of integrin at Y759 may serve to facilitate the reorganization of the integrin–cytoskeleton signaling complex during platelet spreading.


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)

Localization of calpain-cleaved β3 in spreading platelets. Platelets in Tyrode's buffer (1 × 108/ml) were incubated in the chamber slides precoated with 20 μg/ml fibrinogen for 2 h at 37°C. After three washes, the adherent platelets were fixed with 4% paraformaldehyde and permeabilized. The slides were incubated subsequently with Mab15, specific for integrin β3 extracellular domain (green), and with one of the cleavage site–specific antibodies (Ab759 and Ab754) or an antibody against the COOH terminus of β3 (Ab762) (red), and then with Alexa Fluor 488–conjugated anti–mouse IgG antibody and Alexa Fluor 546–conjugated anti–rabbit IgG antibodies. The slides were scanned under a confocal microscope (63× lens). Images of the green and red fluorescence overlay are shown in the left column. Green fluorescence (Mab15) from the same images is shown in the middle column. Red fluorescence (cleavage site–specific antibodies) from the same images is shown in the right column.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172800&req=5

fig8: Localization of calpain-cleaved β3 in spreading platelets. Platelets in Tyrode's buffer (1 × 108/ml) were incubated in the chamber slides precoated with 20 μg/ml fibrinogen for 2 h at 37°C. After three washes, the adherent platelets were fixed with 4% paraformaldehyde and permeabilized. The slides were incubated subsequently with Mab15, specific for integrin β3 extracellular domain (green), and with one of the cleavage site–specific antibodies (Ab759 and Ab754) or an antibody against the COOH terminus of β3 (Ab762) (red), and then with Alexa Fluor 488–conjugated anti–mouse IgG antibody and Alexa Fluor 546–conjugated anti–rabbit IgG antibodies. The slides were scanned under a confocal microscope (63× lens). Images of the green and red fluorescence overlay are shown in the left column. Green fluorescence (Mab15) from the same images is shown in the middle column. Red fluorescence (cleavage site–specific antibodies) from the same images is shown in the right column.
Mentions: Calpain only cleaves a percentage of β3 molecules in platelets stimulated with thrombin, suggesting that cleavage of β3 is likely to have only a localized effect on integrin function. It is known that during cell spreading on integrin ligands, integrin forms a localized signaling complex with cytoskeletal and signaling molecules that is dynamically regulated and involves calpain activity (Bialkowska et al., 2000). To determine if selective integrin cleavage occurred at specific regions during platelet spreading on integrin ligands, platelets were allowed to spread on fibrinogen for 60 min and then double stained with calpain cleavage–specific antibodies and a monoclonal antibody against an extracellular epitope of β3 that is not affected by calpain cleavage. Consistent with the above results in thrombin-stimulated platelets, calpain cleavage occurred mainly at Y759 (Fig. 8), but cleavages at F754 (Fig. 8) or the more NH2-terminal sites (unpublished data) were significantly weaker. Whereas the β3 molecules were distributed throughout the spreading platelets with particularly strong staining at the edges, calpain-cleaved β3 was not seen at the edges (or in the pseudopods) but was concentrated in the central region of the spreading platelets as clusters. As integrin engagement with immobilized ligands starts from the central region where discoid platelets initially adhere, our data suggest that cleavage of β3 by calpain occurred only to the population of the integrin that is no longer at the leading edge of spreading platelets. This selective cleavage of the centrally located integrin population (mainly at Y759) may serve to down-regulate local integrin outside-in signaling in these areas without affecting the activation state of the integrin. It is possible that selective cleavage of integrin at Y759 may serve to facilitate the reorganization of the integrin–cytoskeleton signaling complex during platelet spreading.

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