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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 fibrinogen binding to wild type or various mutants of αIIbβ3. (A) Cells (123, 1b9/741, 1b9/747, 1b9/754, 1b9/759, and 1b9/Y759A) suspended in modified Tyrode's buffer were incubated at 22°C for 30 min with Oregon green–labeled fibrinogen (30 μg/ml) and 1 mg/ml of ristocetin with (right) or without (left) adding 25 μg/ml of vWF, and then were analyzed for fluorescence intensity by flow cytometry (solid lines). Nonspecific binding of fibrinogen was estimated by adding 1 mM RGDS (shaded lines). (B) Quantitative results from three experiments (means ± SD) are expressed as fibrinogen binding index (total bound fluorescence intensity/nonspecifically bound fluorescence intensity).
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fig3: vWF-induced fibrinogen binding to wild type or various mutants of αIIbβ3. (A) Cells (123, 1b9/741, 1b9/747, 1b9/754, 1b9/759, and 1b9/Y759A) suspended in modified Tyrode's buffer were incubated at 22°C for 30 min with Oregon green–labeled fibrinogen (30 μg/ml) and 1 mg/ml of ristocetin with (right) or without (left) adding 25 μg/ml of vWF, and then were analyzed for fluorescence intensity by flow cytometry (solid lines). Nonspecific binding of fibrinogen was estimated by adding 1 mM RGDS (shaded lines). (B) Quantitative results from three experiments (means ± SD) are expressed as fibrinogen binding index (total bound fluorescence intensity/nonspecifically bound fluorescence intensity).

Mentions: To understand the roles of the COOH-terminal region of β3 in inside-out signaling of αIIbβ3, we examined vWF-induced fibrinogen binding to cell lines expressing GPIb-IX and truncation mutants of αIIbβ3 (1b9/741, 1b9/747, 1b9/754, and 1b9/759). Fig. 3 shows that truncation of the β3 cytoplasmic domain at the site COOH terminal to Y759 did not affect vWF-induced activation of fibrinogen binding to integrin αIIbβ3. In contrast, truncations at F754, Y747, or T741 abolished vWF-induced integrin activation. Thus, it appears that the RGT sequence at the COOH terminus of the β3 cytoplasmic domain is not required for integrin activation, whereas the sequence between F754 and Y759, which contains an NXXY motif, is required for integrin activation. To further determine if the NXXY motif is important in integrin activation, we examined vWF-induced fibrinogen binding to CHO cells coexpressing GPIb-IX and the Y759A mutant of integrin αIIbβ3. Indeed, vWF-induced fibrinogen binding was abolished in the Y759A mutant, indicating that the NXXY motif, particularly Y759, is required for vWF-induced activation of 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 fibrinogen binding to wild type or various mutants of αIIbβ3. (A) Cells (123, 1b9/741, 1b9/747, 1b9/754, 1b9/759, and 1b9/Y759A) suspended in modified Tyrode's buffer were incubated at 22°C for 30 min with Oregon green–labeled fibrinogen (30 μg/ml) and 1 mg/ml of ristocetin with (right) or without (left) adding 25 μg/ml of vWF, and then were analyzed for fluorescence intensity by flow cytometry (solid lines). Nonspecific binding of fibrinogen was estimated by adding 1 mM RGDS (shaded lines). (B) Quantitative results from three experiments (means ± SD) are expressed as fibrinogen binding index (total bound fluorescence intensity/nonspecifically bound fluorescence intensity).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172800&req=5

fig3: vWF-induced fibrinogen binding to wild type or various mutants of αIIbβ3. (A) Cells (123, 1b9/741, 1b9/747, 1b9/754, 1b9/759, and 1b9/Y759A) suspended in modified Tyrode's buffer were incubated at 22°C for 30 min with Oregon green–labeled fibrinogen (30 μg/ml) and 1 mg/ml of ristocetin with (right) or without (left) adding 25 μg/ml of vWF, and then were analyzed for fluorescence intensity by flow cytometry (solid lines). Nonspecific binding of fibrinogen was estimated by adding 1 mM RGDS (shaded lines). (B) Quantitative results from three experiments (means ± SD) are expressed as fibrinogen binding index (total bound fluorescence intensity/nonspecifically bound fluorescence intensity).
Mentions: To understand the roles of the COOH-terminal region of β3 in inside-out signaling of αIIbβ3, we examined vWF-induced fibrinogen binding to cell lines expressing GPIb-IX and truncation mutants of αIIbβ3 (1b9/741, 1b9/747, 1b9/754, and 1b9/759). Fig. 3 shows that truncation of the β3 cytoplasmic domain at the site COOH terminal to Y759 did not affect vWF-induced activation of fibrinogen binding to integrin αIIbβ3. In contrast, truncations at F754, Y747, or T741 abolished vWF-induced integrin activation. Thus, it appears that the RGT sequence at the COOH terminus of the β3 cytoplasmic domain is not required for integrin activation, whereas the sequence between F754 and Y759, which contains an NXXY motif, is required for integrin activation. To further determine if the NXXY motif is important in integrin activation, we examined vWF-induced fibrinogen binding to CHO cells coexpressing GPIb-IX and the Y759A mutant of integrin αIIbβ3. Indeed, vWF-induced fibrinogen binding was abolished in the Y759A mutant, indicating that the NXXY motif, particularly Y759, is required for vWF-induced activation of 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