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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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Fibrinogen binding to mutants of αIIbβ3 pretreated with RGDS peptide. Cells coexpressing GPIb-IX and wild-type αIIbβ3 (123) or integrin mutants were preincubated with or without 1 mM of RGDS peptide for 10 min and then fixed with paraformaldehyde (1% in PBS) for 30 min at 22°C. As a negative control, cells expressing GPIb-IX alone (1b9) were identically treated. The cells were washed, resuspended in modified Tyrode's buffer at a concentration of 5 × 105/ml, and then incubated with Oregon green–labeled fibrinogen for 30 min. Fibrinogen binding was detected by flow cytometry. Nonspecific binding was estimated by adding RGDS peptide to the assay. Specific fibrinogen binding was determined by subtracting the geometric means of fluorescence intensity of the nonspecific binding from that of total binding. Shown in the figure are the data (mean ± SD) from three experiments.
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fig4: Fibrinogen binding to mutants of αIIbβ3 pretreated with RGDS peptide. Cells coexpressing GPIb-IX and wild-type αIIbβ3 (123) or integrin mutants were preincubated with or without 1 mM of RGDS peptide for 10 min and then fixed with paraformaldehyde (1% in PBS) for 30 min at 22°C. As a negative control, cells expressing GPIb-IX alone (1b9) were identically treated. The cells were washed, resuspended in modified Tyrode's buffer at a concentration of 5 × 105/ml, and then incubated with Oregon green–labeled fibrinogen for 30 min. Fibrinogen binding was detected by flow cytometry. Nonspecific binding was estimated by adding RGDS peptide to the assay. Specific fibrinogen binding was determined by subtracting the geometric means of fluorescence intensity of the nonspecific binding from that of total binding. Shown in the figure are the data (mean ± SD) from three experiments.

Mentions: A possible alternative interpretation to the above results is that the activation-defective mutants of αIIbβ3 (754, 747, and 741) may have a loss of function in the extracellular ligand-binding site. To exclude this possibility, we examined the fibrinogen-binding function of these mutants. It has been demonstrated previously (Du et al., 1991) that binding of the ligand mimetic peptide, RGDS, to the extracellular ligand-binding domain of αIIbβ3 transforms the integrin into an active conformation. After fixation and removal of RGDS, the activated integrin is able to bind fibrinogen. Thus, we examined RGDS-induced fibrinogen binding to cells expressing mutants of αIIbβ3 (Fig. 4). All of the above mutant cell lines bound fibrinogen in a manner similar to wild-type integrin αIIbβ3 (123 cells). In contrast, 1b9 cells, in which no integrin αIIbβ3 was expressed, did not bind fibrinogen after RGDS treatment. These data suggest that the activation-defective mutants used in this study retained fibrinogen-binding function. Therefore, the inability of these mutants to bind fibrinogen after vWF stimulation results from a deficiency in inside-out signaling.


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)

Fibrinogen binding to mutants of αIIbβ3 pretreated with RGDS peptide. Cells coexpressing GPIb-IX and wild-type αIIbβ3 (123) or integrin mutants were preincubated with or without 1 mM of RGDS peptide for 10 min and then fixed with paraformaldehyde (1% in PBS) for 30 min at 22°C. As a negative control, cells expressing GPIb-IX alone (1b9) were identically treated. The cells were washed, resuspended in modified Tyrode's buffer at a concentration of 5 × 105/ml, and then incubated with Oregon green–labeled fibrinogen for 30 min. Fibrinogen binding was detected by flow cytometry. Nonspecific binding was estimated by adding RGDS peptide to the assay. Specific fibrinogen binding was determined by subtracting the geometric means of fluorescence intensity of the nonspecific binding from that of total binding. Shown in the figure are the data (mean ± SD) from three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172800&req=5

fig4: Fibrinogen binding to mutants of αIIbβ3 pretreated with RGDS peptide. Cells coexpressing GPIb-IX and wild-type αIIbβ3 (123) or integrin mutants were preincubated with or without 1 mM of RGDS peptide for 10 min and then fixed with paraformaldehyde (1% in PBS) for 30 min at 22°C. As a negative control, cells expressing GPIb-IX alone (1b9) were identically treated. The cells were washed, resuspended in modified Tyrode's buffer at a concentration of 5 × 105/ml, and then incubated with Oregon green–labeled fibrinogen for 30 min. Fibrinogen binding was detected by flow cytometry. Nonspecific binding was estimated by adding RGDS peptide to the assay. Specific fibrinogen binding was determined by subtracting the geometric means of fluorescence intensity of the nonspecific binding from that of total binding. Shown in the figure are the data (mean ± SD) from three experiments.
Mentions: A possible alternative interpretation to the above results is that the activation-defective mutants of αIIbβ3 (754, 747, and 741) may have a loss of function in the extracellular ligand-binding site. To exclude this possibility, we examined the fibrinogen-binding function of these mutants. It has been demonstrated previously (Du et al., 1991) that binding of the ligand mimetic peptide, RGDS, to the extracellular ligand-binding domain of αIIbβ3 transforms the integrin into an active conformation. After fixation and removal of RGDS, the activated integrin is able to bind fibrinogen. Thus, we examined RGDS-induced fibrinogen binding to cells expressing mutants of αIIbβ3 (Fig. 4). All of the above mutant cell lines bound fibrinogen in a manner similar to wild-type integrin αIIbβ3 (123 cells). In contrast, 1b9 cells, in which no integrin αIIbβ3 was expressed, did not bind fibrinogen after RGDS treatment. These data suggest that the activation-defective mutants used in this study retained fibrinogen-binding function. Therefore, the inability of these mutants to bind fibrinogen after vWF stimulation results from a deficiency in inside-out signaling.

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