Roles of integrin β3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model

doi:10.1007/s11684-016-0460-0

Front. Med.

2016, Vol. 10

Issue (3) : 311-319 https://doi.org/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 Huang^1,²,Yulan Zhou^1,³,Xiaoyu Su¹,Yuanjing Lyu¹,Lanlan Tao¹,Xiaofeng Shi^1,⁴,Ping Liu^1,⁵,Zhangbiao Long^1,⁶,Zheng Ruan¹,Bing Xiao¹,Wenda Xi⁷,Quansheng Zhou⁸,Jianhua Mao^1,^*(

),Xiaodong Xi^1,^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 Author(s): Jianhua Mao,Xiaodong Xi

Just Accepted Date: 08 July 2016 Online First Date: 10 August 2016 Issue Date: 30 August 2016

Cite this article:

Jiansong Huang,Yulan Zhou,Xiaoyu Su, et al. Roles of integrin β3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model[J]. Front. Med., 2016, 10(3): 311-319.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0460-0
https://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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