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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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vWF-induced activation of fibrinogen binding to cell lines coexpressing GPIb-IX and αIIbβ3 reflects specific activation of integrin αIIbβ3. (A) CHO cells (CHO), CHO cells coexpressing GPIb-IX and integrin αIIbβ3 (123), and CHO cells coexpressing GPIb-IX and β3 in the absence of αIIb (1b9/β3) were incubated with control mouse IgG, αIIbβ3-specific antibodies, 2G12 and D57, and an antibody recognizing integrin αvβ3, anti-VNR1. The reactivity of these antibodies was detected by flow cytometry. (B) 123 cells were preincubated with 100 μg/ml of control mouse IgG, 2G12, or anti-VNR1 (previously shown to inhibit ligand-binding functions of αIIbβ3 or αvβ3, respectively) for 10 min at 22°C, and then Oregon green–labeled fibrinogen (30 μg/ml) was added and activated with vWF in the presence of ristocetin. Nonspecific binding was estimated by adding the integrin inhibitor RGDS. Quantitative results from three experiments (means ± SD) are expressed as fibrinogen binding index (total bound fibrinogen/nonspecifically bound fibrinogen in the presence of RGDS). Statistical differences between samples were analyzed using the RRISM software and the Repeated Measures ANOVA method.
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fig2: vWF-induced activation of fibrinogen binding to cell lines coexpressing GPIb-IX and αIIbβ3 reflects specific activation of integrin αIIbβ3. (A) CHO cells (CHO), CHO cells coexpressing GPIb-IX and integrin αIIbβ3 (123), and CHO cells coexpressing GPIb-IX and β3 in the absence of αIIb (1b9/β3) were incubated with control mouse IgG, αIIbβ3-specific antibodies, 2G12 and D57, and an antibody recognizing integrin αvβ3, anti-VNR1. The reactivity of these antibodies was detected by flow cytometry. (B) 123 cells were preincubated with 100 μg/ml of control mouse IgG, 2G12, or anti-VNR1 (previously shown to inhibit ligand-binding functions of αIIbβ3 or αvβ3, respectively) for 10 min at 22°C, and then Oregon green–labeled fibrinogen (30 μg/ml) was added and activated with vWF in the presence of ristocetin. Nonspecific binding was estimated by adding the integrin inhibitor RGDS. Quantitative results from three experiments (means ± SD) are expressed as fibrinogen binding index (total bound fibrinogen/nonspecifically bound fibrinogen in the presence of RGDS). Statistical differences between samples were analyzed using the RRISM software and the Repeated Measures ANOVA method.

Mentions: We recently reported the reconstitution of GPIb-IX–mediated integrin activation in CHO cells expressing recombinant human GPIb-IX and integrin αIIbβ3 (123 cells) (Gu et al., 1999). In this reconstituted integrin activation model, vWF binding to GPIb-IX in the presence of ristocetin activates fibrinogen binding to the 123 cells in an RGDS-dependent manner, thus allowing the study of integrin inside-out signaling using a specific recombinant DNA approach. Although β3-transfected CHO cells also express an αvβ3 integrin composed of endogenous αv and recombinant β3, Fig. 2 shows that vWF-induced fibrinogen binding to 123 cells is specifically inhibited by a monoclonal antibody that recognizes human αIIbβ3, but not by a blocking monoclonal antibody that recognizes αvβ3. Also, a cell line expressing GPIb-IX and β3 without αIIb failed to show RGDS-dependent fibrinogen binding under identical conditions (unpublished data). These results indicate that vWF-induced fibrinogen binding is mediated by αIIbβ3 but not αvβ3. Thus, vWF-induced fibrinogen binding to 123 cells specifically reflects the activation of the platelet integrin αIIbβ3.


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)

vWF-induced activation of fibrinogen binding to cell lines coexpressing GPIb-IX and αIIbβ3 reflects specific activation of integrin αIIbβ3. (A) CHO cells (CHO), CHO cells coexpressing GPIb-IX and integrin αIIbβ3 (123), and CHO cells coexpressing GPIb-IX and β3 in the absence of αIIb (1b9/β3) were incubated with control mouse IgG, αIIbβ3-specific antibodies, 2G12 and D57, and an antibody recognizing integrin αvβ3, anti-VNR1. The reactivity of these antibodies was detected by flow cytometry. (B) 123 cells were preincubated with 100 μg/ml of control mouse IgG, 2G12, or anti-VNR1 (previously shown to inhibit ligand-binding functions of αIIbβ3 or αvβ3, respectively) for 10 min at 22°C, and then Oregon green–labeled fibrinogen (30 μg/ml) was added and activated with vWF in the presence of ristocetin. Nonspecific binding was estimated by adding the integrin inhibitor RGDS. Quantitative results from three experiments (means ± SD) are expressed as fibrinogen binding index (total bound fibrinogen/nonspecifically bound fibrinogen in the presence of RGDS). Statistical differences between samples were analyzed using the RRISM software and the Repeated Measures ANOVA method.
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Related In: Results  -  Collection

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fig2: vWF-induced activation of fibrinogen binding to cell lines coexpressing GPIb-IX and αIIbβ3 reflects specific activation of integrin αIIbβ3. (A) CHO cells (CHO), CHO cells coexpressing GPIb-IX and integrin αIIbβ3 (123), and CHO cells coexpressing GPIb-IX and β3 in the absence of αIIb (1b9/β3) were incubated with control mouse IgG, αIIbβ3-specific antibodies, 2G12 and D57, and an antibody recognizing integrin αvβ3, anti-VNR1. The reactivity of these antibodies was detected by flow cytometry. (B) 123 cells were preincubated with 100 μg/ml of control mouse IgG, 2G12, or anti-VNR1 (previously shown to inhibit ligand-binding functions of αIIbβ3 or αvβ3, respectively) for 10 min at 22°C, and then Oregon green–labeled fibrinogen (30 μg/ml) was added and activated with vWF in the presence of ristocetin. Nonspecific binding was estimated by adding the integrin inhibitor RGDS. Quantitative results from three experiments (means ± SD) are expressed as fibrinogen binding index (total bound fibrinogen/nonspecifically bound fibrinogen in the presence of RGDS). Statistical differences between samples were analyzed using the RRISM software and the Repeated Measures ANOVA method.
Mentions: We recently reported the reconstitution of GPIb-IX–mediated integrin activation in CHO cells expressing recombinant human GPIb-IX and integrin αIIbβ3 (123 cells) (Gu et al., 1999). In this reconstituted integrin activation model, vWF binding to GPIb-IX in the presence of ristocetin activates fibrinogen binding to the 123 cells in an RGDS-dependent manner, thus allowing the study of integrin inside-out signaling using a specific recombinant DNA approach. Although β3-transfected CHO cells also express an αvβ3 integrin composed of endogenous αv and recombinant β3, Fig. 2 shows that vWF-induced fibrinogen binding to 123 cells is specifically inhibited by a monoclonal antibody that recognizes human αIIbβ3, but not by a blocking monoclonal antibody that recognizes αvβ3. Also, a cell line expressing GPIb-IX and β3 without αIIb failed to show RGDS-dependent fibrinogen binding under identical conditions (unpublished data). These results indicate that vWF-induced fibrinogen binding is mediated by αIIbβ3 but not αvβ3. Thus, vWF-induced fibrinogen binding to 123 cells specifically reflects the activation of the platelet integrin αIIbβ3.

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