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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (3) : 311-319     DOI: 10.1007/s11684-016-0460-0
RESEARCH ARTICLE |
Roles of integrin β3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model
Jiansong Huang1,2,Yulan Zhou1,3,Xiaoyu Su1,Yuanjing Lyu1,Lanlan Tao1,Xiaofeng Shi1,4,Ping Liu1,5,Zhangbiao Long1,6,Zheng Ruan1,Bing Xiao1,Wenda Xi7,Quansheng Zhou8,Jianhua Mao1,*(),Xiaodong Xi1,9,*()
1. State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2. Institute of Hematology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
3. Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China
4. Department of Hematology, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China
5. Department of Pediatrics, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200001, China
6. Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100073, China
7. Shanghai Institute of Hypertension, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
8. Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, 2011 Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215123, China
9. Sino-French Research Centre for Life Sciences and Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
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Abstract  

We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins (i.e., Tac-β3, Tac-β3D741, Tac-β3D747, Tac-β3D754, Tac-β3D759, and Tac-β3DNITY) consisting of the extracellular and transmembrane domains of human IL-2 receptor (Tac) and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin αIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3DNITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3D759, Tac-β3DNITY, and Tac-β3D754, but not Tac-β3D747 or Tac-β3D741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3 and Tac-β3DNITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3D759 and 123/Tac-β3D754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3D747 and 123/Tac-β3D741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.

Keywords integrin β3      signal transduction      trans-dominant inhibition model     
Corresponding Authors: Jianhua Mao,Xiaodong Xi   
Just Accepted Date: 08 July 2016   Online First Date: 10 August 2016    Issue Date: 30 August 2016
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0460-0     OR     http://academic.hep.com.cn/fmd/EN/Y2016/V10/I3/311
Fig.1  Chimeric Tac-β3 or truncation mutants of Tac-β3 expressed in the 123 CHO cells. (A) The amino acid sequence for integrin β3 cytoplasmic tail or truncation mutants (β3D759, β3DNITY, β3D754, β3D747, and β3D741) of Tac-β3. (B) Flow cytometric analysis of the expression of extracellular αIIb, β3, glycoprotein GPIb-IX, and Tac. The expression of integrin αIIb and glycoprotein GPIb-IX was tested by SZ-22 and SZ-2, respectively. The expression level of Tac-β3 chimeras and β3 was examined by flow cytometry with antibodies specific for Tac (7G7B6) and for the extracellular domain of the β3 subunit (SZ-21), respectively. Non-specific mouse IgG was used as a negative control. The lateral axis represents the fluorescence intensity, and the vertical axis indicates the cell count. (C) Western blot analysis of the expression level of the chimeric Tac-β3 in the 123 CHO cells. The 123 CHO cells with or without chimeric Tac-β3 were lysed and blotted for the C3a antibody, which recognizes the β3 cytoplasmic tail. The Tac-β3 expression level is stronger than that of β3. Actin was used as a loading control.
Fig.2  Effects of Tac-chimeras on integrin-dependent cell adhesion and spreading on immobilized fibrinogen. (A) The 123 CHO cells expressing differently truncated Tac-chimeras were allowed to adhere on fibrinogen-coated coverslips for 2 h. The morphology of the transfected 123 CHO cell clones was examined under an inverted microscope. The representative images from one of the three experiments with similar results are displayed. The scale bar represents 40 mm. (B) Quantitative analysis of the adhesive phenotype of the transfected 123 CHO cell clones plated on immobilized fibrinogen (100 cells counted, n = 3, mean±SD). (C) The percentage of the surface areas covered by the CHO cells. **P<0.05, *P<0.01, compared with the 123 CHO cells. (D) The adherent CHO cells after washing were quantified by PNPP assay. **P<0.05, *P<0.01, compared with the 123 CHO cells.
Fig.3  Binding of soluble fibrinogen to the 123 CHO cells expressing different Tac-chimeras. (A) Binding of Alexa Fluor 488-conjugated fibrinogen (100 mg/ml) to the CHO cells was measured by flow cytometry after the addition of ristocetin and vWF. The lateral and vertical axis represents the fluorescence intensity and the cell count, respectively. (B) The fluorescence intensity calculated from the data in panel A is presented as the mean and SD of three independent experiments. **P<0.05, *P<0.01, compared with the 123 CHO cells.
Fig.4  Sequence of the integrin β3 tail and the interaction sites with cytoplasmic signaling proteins. The mapped positions indicate the sites, where the integrin β3 cytoplasmic tail enables the interaction with cytoplasmic signaling proteins. The sites of the calpain cleavage are indicated by arrows, meanwhile the amino acid residues that can be phosphorylated in integrin αIIbβ3 signaling are labeled in red.
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