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

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-β3△741, Tac-β3△747, Tac-β3△754, Tac-β3△759, and Tac-β3ANITY） 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 uIIbβ3. 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-β3△NITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3△759, Tac-β3ANITY, and Tac-β3△754, but not Tac-β3△747 or Tac-β3△741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3△ and Tac-β3ANITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3△759 and 123/Tac-β3△754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3△747 and 123/Tac-β3△741 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.