293T细胞模型中整合素β3胞浆尾段不同氨基酸基序对αⅡbβ3介导的细胞功能的影响
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摘要
目的:建立含有Calpain切割相关的β3胞浆尾段突变体的293T细胞模型,研究β3胞浆尾段不同氨基酸基序对αⅡbβ3介导的细胞功能的影响。方法:利用293T细胞模型建立共表达人类野生型整合素αⅡb和野生型β3或突变型β3(β3ΔNITY(β3胞浆段截去NITY基序)、β3Δ754(β3胞浆段截去羧基末端最后8个氨基酸TNITYRGT)、β3Δ759(β3胞浆段截去羧基末端最后3个氨基酸RGT)的稳转细胞株,通过细胞在固相化纤维蛋白原上的黏附及伸展试验检测各稳转细胞株的黏附及伸展功能。结果:成功建立了293T-αⅡbβ3ΔNITY、293T-αⅡbβ3Δ754、293T-αⅡbβ3Δ759、293T-αⅡbβ3细胞株,稳定表达野生型整合素β3全长的293T--αⅡbβ3细胞株较293T细胞具有良好的在固相化纤维蛋白原上的黏附和伸展能力,可以作为血小板的替代模型,而293T-αⅡbβ3ΔNITY稳转细胞株的黏附和伸展能力稍低于293T-αⅡbβ3稳转细胞株,但293T-αⅡbβ3Δ754细胞、293T-αⅡbβ3Δ759细胞株的黏附和伸展能力均明显受损。结论:对整合素β3介导的细胞伸展功能,RGT基序至关重要,而NITY并非必不可少,同时所建β3胞浆尾段不同突变体的293T细胞株也为进一步的质谱分析数据库比对奠定了基础。
Objective: To establish 293 T cell lines stably expressing Calpain-cleavage related β3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function. Methods: 293 T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY(β3 cytoplasmic tail NITY motif deleted), β3-Δ754(β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759(β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. Results: 293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293 T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen. Conclusion: To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293 T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.
Objective: To establish 293 T cell lines stably expressing Calpain-cleavage related β3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function. Methods: 293 T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY(β3 cytoplasmic tail NITY motif deleted), β3-Δ754(β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759(β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. Results: 293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293 T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen. Conclusion: To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293 T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.
引文
1 Hynes RO.Integrins:bidirectional,allosteric signaling machines.Cell,2002;110(6):673-687.
2 Ussar S,Wang HV,Linder S,et al.The Kindlins:subcellular localization and expression during murine development.Exp Cell Res,2006;312(16):3142-3151.
3 Schurpf T,Springer TA.Regulation of integrin affinity on cell surfaces.EMBO J,2011;30(23):4712-4727.
4 Coughlin SR.Protease-activated receptors in hemostasis,thrombosis and vascular biology.J Thromb Haemost,2005;3(8):1800-1814.
5 Tao L,Zhang Y,Xi X,et al.Recent advances in the understanding of the molecular mechanisms regulating platelet integrin alphaIIbbeta3 activation.Protein Cell,2010;1(7):627-637.
6 Xi X,Bodnar RJ,Li Z,et al.Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling.J Cell Biol,2003;162(2):329-339.
7 Xi X,Flevaris P,Stojanovic A,et al.Tyrosine phosphorylation of the integrin beta 3 subunit regulates beta 3 cleavage by calpain.JBiol Chem,2006;281(40):29426-29430.
8 Klockenbusch C,Walsh GM,Brown LM,et al.Global proteome analysis identifies active immunoproteasome subunits in human platelets.Mol Cell Proteomics,2014;13(12):3308-3319.
9 Zou ZY,Chen H,Schmaier AA,et al.Structure-function analysis reveals discrete beta3 integrin inside-out and outside-in signaling pathways in platelets.Blood,2007;109(8):3284-3290.
10 O'Toole TE,Loftus JC,Du XP,et al.Affinity modulation of the alpha IIb beta 3 integrin(platelet GPIIb-IIIa)is an intrinsic property of the receptor.Cell Regul,1990;1(12):883-893.
11 O'Toole TE,Mandelman D,Forsyth J,et al.Modulation of the affinity of integrin alpha IIb beta 3(GPIIb-IIIa)by the cytoplasmic domain of alpha IIb.Science,1991;254(5033):845-847.
12 Shi X,Yang J,Cui X,et al.Functional effect of the mutations similar to the cleavage during platelet activation at integrin beta3cytoplasmic Tail when expressed in mouse platelets.PLoS One,2016;11(11):e0166136.
13 Su X,Mi J,Yan J,et al.RGT,a synthetic peptide corresponding to the integrin beta 3 cytoplasmic C-terminal sequence,selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin alpha IIb beta 3 with Src kinase.Blood,2008;112(3):592-602.
2 Ussar S,Wang HV,Linder S,et al.The Kindlins:subcellular localization and expression during murine development.Exp Cell Res,2006;312(16):3142-3151.
3 Schurpf T,Springer TA.Regulation of integrin affinity on cell surfaces.EMBO J,2011;30(23):4712-4727.
4 Coughlin SR.Protease-activated receptors in hemostasis,thrombosis and vascular biology.J Thromb Haemost,2005;3(8):1800-1814.
5 Tao L,Zhang Y,Xi X,et al.Recent advances in the understanding of the molecular mechanisms regulating platelet integrin alphaIIbbeta3 activation.Protein Cell,2010;1(7):627-637.
6 Xi X,Bodnar RJ,Li Z,et al.Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling.J Cell Biol,2003;162(2):329-339.
7 Xi X,Flevaris P,Stojanovic A,et al.Tyrosine phosphorylation of the integrin beta 3 subunit regulates beta 3 cleavage by calpain.JBiol Chem,2006;281(40):29426-29430.
8 Klockenbusch C,Walsh GM,Brown LM,et al.Global proteome analysis identifies active immunoproteasome subunits in human platelets.Mol Cell Proteomics,2014;13(12):3308-3319.
9 Zou ZY,Chen H,Schmaier AA,et al.Structure-function analysis reveals discrete beta3 integrin inside-out and outside-in signaling pathways in platelets.Blood,2007;109(8):3284-3290.
10 O'Toole TE,Loftus JC,Du XP,et al.Affinity modulation of the alpha IIb beta 3 integrin(platelet GPIIb-IIIa)is an intrinsic property of the receptor.Cell Regul,1990;1(12):883-893.
11 O'Toole TE,Mandelman D,Forsyth J,et al.Modulation of the affinity of integrin alpha IIb beta 3(GPIIb-IIIa)by the cytoplasmic domain of alpha IIb.Science,1991;254(5033):845-847.
12 Shi X,Yang J,Cui X,et al.Functional effect of the mutations similar to the cleavage during platelet activation at integrin beta3cytoplasmic Tail when expressed in mouse platelets.PLoS One,2016;11(11):e0166136.
13 Su X,Mi J,Yan J,et al.RGT,a synthetic peptide corresponding to the integrin beta 3 cytoplasmic C-terminal sequence,selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin alpha IIb beta 3 with Src kinase.Blood,2008;112(3):592-602.