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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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Effects of mutations on integrin-dependent stable cell adhesion and spreading on vWF or fibrinogen (Fg) under static conditions. The indicated cell lines in modified Tyrode's buffer (106/ml) were allowed to incubate in fibrinogen- or vWF-coated microtiter wells at 37°C for 1 h. The wells were washed three times, and the cells remaining adherent were quantitated using an acid phosphatase assay, as described under the Materials and methods (A), and photographed to document cell spreading (B). The means and standard deviations of triplicate samples are shown in A.
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fig5: Effects of mutations on integrin-dependent stable cell adhesion and spreading on vWF or fibrinogen (Fg) under static conditions. The indicated cell lines in modified Tyrode's buffer (106/ml) were allowed to incubate in fibrinogen- or vWF-coated microtiter wells at 37°C for 1 h. The wells were washed three times, and the cells remaining adherent were quantitated using an acid phosphatase assay, as described under the Materials and methods (A), and photographed to document cell spreading (B). The means and standard deviations of triplicate samples are shown in A.

Mentions: An important function for αIIbβ3 is to mediate stable platelet adhesion and spreading on immobilized integrin ligands such as vWF and fibrinogen. Integrin-dependent stable cell adhesion and spreading on vWF are significantly enhanced by inside-out signaling induced by GPIb-IX–vWF interaction (Savage et al., 1992) and also require integrin outside-in signaling. Integrin αIIbβ3–mediated stable cell adhesion and spreading to immobilized fibrinogen, however, do not require inside-out signaling (Coller, 1980; Savage et al., 1992) but are dependent upon ligand-induced integrin activation and outside-in signaling (Du et al., 1991; Phillips et al., 1991; Ginsberg et al., 1995; Shattil et al., 1998). Thus, to determine the roles of the COOH-terminal region of β3 in outside-in and inside-out signaling, we examined stable adhesion and spreading of the mutant cell lines to fibrinogen and vWF. As previously reported (Gu et al., 1999), CHO cells (without transfected GPIb-IX and integrin αIIbβ3) poorly adhere to vWF or fibrinogen. CHO cells expressing GPIb-IX alone also poorly adhere to both vWF and fibrinogen (Fig. 5). Even in the presence of botrocetin, which enhances the GPIb-IX binding affinity of vWF and allows GPIb-IX–dependent stable cell adhesion to vWF, these cells poorly spread on vWF (unpublished data), indicating that β3 integrin is required for cell spreading on vWF (Gu et al., 1999). As expected, CHO cells expressing integrin αIIbβ3 (2b3a) showed high levels of adhesion and spreading on fibrinogen but poorly adhered to vWF within the first hour of incubation (Fig. 5). However, with prolonged incubation, adhesion of 2b3a cells to vWF was slowly increased (unpublished data), suggesting a low affinity interaction between β3 integrins and vWF. Coexpression of GPIb-IX with αIIbβ3 (123 cells) significantly enhanced and accelerated integrin-dependent cell adhesion and spreading on vWF, indicating that GPIb-IX activates integrin, leading to integrin-dependent cell spreading and stable adhesion. Cleavage of β3 at or NH2 terminal to F754 inhibited integrin-dependent stable cell adhesion and spreading on vWF and fibrinogen (Fig. 5), which is consistent with previous findings that this region of the integrin cytoplasmic domain is important for adhesion and spreading (Ylanne et al., 1993, 1995). Interestingly, truncation of β3 at Y759 also showed a reduced stable adhesion to both vWF and fibrinogen, and most 1b9/759 cells that adhered to fibrinogen or vWF spread poorly (Fig. 5). Reduced stable adhesion in 1b9/759 cells was not caused by a defect in inside-out signaling, as αIIbβ3-dependent cell adhesion to fibrinogen under these conditions does not require inside-out signaling, and 1b9/759 cells showed normal inside-out signaling, as indicated by soluble fibrinogen binding (Fig. 3). Reduced stable adhesion of 1b9/759 cells is likely resultant from a decreased resistance to the washing procedure in the adhesion assay, as gentler washing can significantly increase 1b9/759 cell adhesion (unpublished data; Fig. 6). Thus, reduced cell adhesion and spreading in 1b9/759 cells suggest that integrin outside-in signaling in this truncation mutant is impaired.


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)

Effects of mutations on integrin-dependent stable cell adhesion and spreading on vWF or fibrinogen (Fg) under static conditions. The indicated cell lines in modified Tyrode's buffer (106/ml) were allowed to incubate in fibrinogen- or vWF-coated microtiter wells at 37°C for 1 h. The wells were washed three times, and the cells remaining adherent were quantitated using an acid phosphatase assay, as described under the Materials and methods (A), and photographed to document cell spreading (B). The means and standard deviations of triplicate samples are shown in A.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172800&req=5

fig5: Effects of mutations on integrin-dependent stable cell adhesion and spreading on vWF or fibrinogen (Fg) under static conditions. The indicated cell lines in modified Tyrode's buffer (106/ml) were allowed to incubate in fibrinogen- or vWF-coated microtiter wells at 37°C for 1 h. The wells were washed three times, and the cells remaining adherent were quantitated using an acid phosphatase assay, as described under the Materials and methods (A), and photographed to document cell spreading (B). The means and standard deviations of triplicate samples are shown in A.
Mentions: An important function for αIIbβ3 is to mediate stable platelet adhesion and spreading on immobilized integrin ligands such as vWF and fibrinogen. Integrin-dependent stable cell adhesion and spreading on vWF are significantly enhanced by inside-out signaling induced by GPIb-IX–vWF interaction (Savage et al., 1992) and also require integrin outside-in signaling. Integrin αIIbβ3–mediated stable cell adhesion and spreading to immobilized fibrinogen, however, do not require inside-out signaling (Coller, 1980; Savage et al., 1992) but are dependent upon ligand-induced integrin activation and outside-in signaling (Du et al., 1991; Phillips et al., 1991; Ginsberg et al., 1995; Shattil et al., 1998). Thus, to determine the roles of the COOH-terminal region of β3 in outside-in and inside-out signaling, we examined stable adhesion and spreading of the mutant cell lines to fibrinogen and vWF. As previously reported (Gu et al., 1999), CHO cells (without transfected GPIb-IX and integrin αIIbβ3) poorly adhere to vWF or fibrinogen. CHO cells expressing GPIb-IX alone also poorly adhere to both vWF and fibrinogen (Fig. 5). Even in the presence of botrocetin, which enhances the GPIb-IX binding affinity of vWF and allows GPIb-IX–dependent stable cell adhesion to vWF, these cells poorly spread on vWF (unpublished data), indicating that β3 integrin is required for cell spreading on vWF (Gu et al., 1999). As expected, CHO cells expressing integrin αIIbβ3 (2b3a) showed high levels of adhesion and spreading on fibrinogen but poorly adhered to vWF within the first hour of incubation (Fig. 5). However, with prolonged incubation, adhesion of 2b3a cells to vWF was slowly increased (unpublished data), suggesting a low affinity interaction between β3 integrins and vWF. Coexpression of GPIb-IX with αIIbβ3 (123 cells) significantly enhanced and accelerated integrin-dependent cell adhesion and spreading on vWF, indicating that GPIb-IX activates integrin, leading to integrin-dependent cell spreading and stable adhesion. Cleavage of β3 at or NH2 terminal to F754 inhibited integrin-dependent stable cell adhesion and spreading on vWF and fibrinogen (Fig. 5), which is consistent with previous findings that this region of the integrin cytoplasmic domain is important for adhesion and spreading (Ylanne et al., 1993, 1995). Interestingly, truncation of β3 at Y759 also showed a reduced stable adhesion to both vWF and fibrinogen, and most 1b9/759 cells that adhered to fibrinogen or vWF spread poorly (Fig. 5). Reduced stable adhesion in 1b9/759 cells was not caused by a defect in inside-out signaling, as αIIbβ3-dependent cell adhesion to fibrinogen under these conditions does not require inside-out signaling, and 1b9/759 cells showed normal inside-out signaling, as indicated by soluble fibrinogen binding (Fig. 3). Reduced stable adhesion of 1b9/759 cells is likely resultant from a decreased resistance to the washing procedure in the adhesion assay, as gentler washing can significantly increase 1b9/759 cell adhesion (unpublished data; Fig. 6). Thus, reduced cell adhesion and spreading in 1b9/759 cells suggest that integrin outside-in signaling in this truncation mutant is impaired.

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