Limits...
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.

Show MeSH

Related in: MedlinePlus

Truncation mutants of αIIbβ3 coexpressed with GPIb-IX in CHO cells. (A) Schematics showing truncation mutants of β3 that mimic calpain cleavage at four previously identified sites. A Y759A point mutation is also depicted. (B) The mutants described in A were cotransfected with αIIb in a CHO cell line already expressing GPIb-IX, and stable cell lines were established. Expression of correct truncation mutants in each of the cell lines was verified by immunoblotting cell lysates with antibodies specifically recognizing calpain-cleaved forms of β3 (Ab 741, Ab 747, Ab 754, and Ab 759) and with an antibody recognizing the COOH terminus of β3 (Ab 762). A cell line expressing wild-type αIIbβ3 and GPIb-IX (123) was also immunoblotted with these antibodies as a control. (C) Levels of surface expression of wild type or mutants of αIIbβ3 were examined by flow cytometry with an antibody recognizing αIIbβ3 complex (D57) (dotted lines), and levels of GPIb-IX expression were examined with an antibody against GPIbα, SZ2 (shaded lines). Nonspecific mouse IgG was used as a negative control (solid lines). Please note that levels of expression of each of these mutants were comparable with 123 cells. Expression of GPIb-IX and integrin was also comparable with a cell line expressing GPIb-IX alone (1b9) and a cell line expressing αIIbβ3 alone (2b3a), respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172800&req=5

fig1: Truncation mutants of αIIbβ3 coexpressed with GPIb-IX in CHO cells. (A) Schematics showing truncation mutants of β3 that mimic calpain cleavage at four previously identified sites. A Y759A point mutation is also depicted. (B) The mutants described in A were cotransfected with αIIb in a CHO cell line already expressing GPIb-IX, and stable cell lines were established. Expression of correct truncation mutants in each of the cell lines was verified by immunoblotting cell lysates with antibodies specifically recognizing calpain-cleaved forms of β3 (Ab 741, Ab 747, Ab 754, and Ab 759) and with an antibody recognizing the COOH terminus of β3 (Ab 762). A cell line expressing wild-type αIIbβ3 and GPIb-IX (123) was also immunoblotted with these antibodies as a control. (C) Levels of surface expression of wild type or mutants of αIIbβ3 were examined by flow cytometry with an antibody recognizing αIIbβ3 complex (D57) (dotted lines), and levels of GPIb-IX expression were examined with an antibody against GPIbα, SZ2 (shaded lines). Nonspecific mouse IgG was used as a negative control (solid lines). Please note that levels of expression of each of these mutants were comparable with 123 cells. Expression of GPIb-IX and integrin was also comparable with a cell line expressing GPIb-IX alone (1b9) and a cell line expressing αIIbβ3 alone (2b3a), respectively.

Mentions: To study the roles of the COOH-terminal region of β3 in integrin signaling, we have generated four truncation mutants of the β3 subunit with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncations at these sites also coincide with previously identified calpain cleavage sites in β3 (Du et al., 1995; Fig. 1 A). These mutants were cotransfected with the integrin αIIb subunit into a CHO cell line that expresses recombinant human GPIb-IX complex (1b9). Complex formation between these β3 mutants and αIIb was verified by flow cytometry using an αIIbβ3 complex–specific monoclonal antibody, D57. Stable cell lines expressing both GPIb-IX and integrin mutants (1b9/741, 1b9/747, 1b9/754, and 1b9/759) were obtained by cell sorting with antibodies specific for integrin αIIbβ3 and GPIb-IX. Expression of correct mutants at each of the four sites was verified by immunoblotting with antibodies that specifically recognize each of these sites only when the site is truncated (Du et al., 1995; Fig. 1 B). In addition, a cell line coexpressing GPIb-IX and a mutant αIIbβ3 bearing alanine substitution of Y759 in β3 was also established (1b9/Y759A). Expression levels of GPIb-IX and integrin αIIbβ3 on the above mutant cell lines were comparable with a cell line expressing GPIb-IX and wild-type integrin αIIbβ3 (123 cells) at the time of experiments (Fig. 1 C).


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)

Truncation mutants of αIIbβ3 coexpressed with GPIb-IX in CHO cells. (A) Schematics showing truncation mutants of β3 that mimic calpain cleavage at four previously identified sites. A Y759A point mutation is also depicted. (B) The mutants described in A were cotransfected with αIIb in a CHO cell line already expressing GPIb-IX, and stable cell lines were established. Expression of correct truncation mutants in each of the cell lines was verified by immunoblotting cell lysates with antibodies specifically recognizing calpain-cleaved forms of β3 (Ab 741, Ab 747, Ab 754, and Ab 759) and with an antibody recognizing the COOH terminus of β3 (Ab 762). A cell line expressing wild-type αIIbβ3 and GPIb-IX (123) was also immunoblotted with these antibodies as a control. (C) Levels of surface expression of wild type or mutants of αIIbβ3 were examined by flow cytometry with an antibody recognizing αIIbβ3 complex (D57) (dotted lines), and levels of GPIb-IX expression were examined with an antibody against GPIbα, SZ2 (shaded lines). Nonspecific mouse IgG was used as a negative control (solid lines). Please note that levels of expression of each of these mutants were comparable with 123 cells. Expression of GPIb-IX and integrin was also comparable with a cell line expressing GPIb-IX alone (1b9) and a cell line expressing αIIbβ3 alone (2b3a), respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172800&req=5

fig1: Truncation mutants of αIIbβ3 coexpressed with GPIb-IX in CHO cells. (A) Schematics showing truncation mutants of β3 that mimic calpain cleavage at four previously identified sites. A Y759A point mutation is also depicted. (B) The mutants described in A were cotransfected with αIIb in a CHO cell line already expressing GPIb-IX, and stable cell lines were established. Expression of correct truncation mutants in each of the cell lines was verified by immunoblotting cell lysates with antibodies specifically recognizing calpain-cleaved forms of β3 (Ab 741, Ab 747, Ab 754, and Ab 759) and with an antibody recognizing the COOH terminus of β3 (Ab 762). A cell line expressing wild-type αIIbβ3 and GPIb-IX (123) was also immunoblotted with these antibodies as a control. (C) Levels of surface expression of wild type or mutants of αIIbβ3 were examined by flow cytometry with an antibody recognizing αIIbβ3 complex (D57) (dotted lines), and levels of GPIb-IX expression were examined with an antibody against GPIbα, SZ2 (shaded lines). Nonspecific mouse IgG was used as a negative control (solid lines). Please note that levels of expression of each of these mutants were comparable with 123 cells. Expression of GPIb-IX and integrin was also comparable with a cell line expressing GPIb-IX alone (1b9) and a cell line expressing αIIbβ3 alone (2b3a), respectively.
Mentions: To study the roles of the COOH-terminal region of β3 in integrin signaling, we have generated four truncation mutants of the β3 subunit with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncations at these sites also coincide with previously identified calpain cleavage sites in β3 (Du et al., 1995; Fig. 1 A). These mutants were cotransfected with the integrin αIIb subunit into a CHO cell line that expresses recombinant human GPIb-IX complex (1b9). Complex formation between these β3 mutants and αIIb was verified by flow cytometry using an αIIbβ3 complex–specific monoclonal antibody, D57. Stable cell lines expressing both GPIb-IX and integrin mutants (1b9/741, 1b9/747, 1b9/754, and 1b9/759) were obtained by cell sorting with antibodies specific for integrin αIIbβ3 and GPIb-IX. Expression of correct mutants at each of the four sites was verified by immunoblotting with antibodies that specifically recognize each of these sites only when the site is truncated (Du et al., 1995; Fig. 1 B). In addition, a cell line coexpressing GPIb-IX and a mutant αIIbβ3 bearing alanine substitution of Y759 in β3 was also established (1b9/Y759A). Expression levels of GPIb-IX and integrin αIIbβ3 on the above mutant cell lines were comparable with a cell line expressing GPIb-IX and wild-type integrin αIIbβ3 (123 cells) at the time of experiments (Fig. 1 C).

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