Analysis of the Regulatory Mechanisms of the RGT Sequence of Integrin β3 Cytoplasmic Tail in Signal Transduction by Using a Dominant Negative Model

Abstract: This study purposed to investigate the molecular mechanisms of the RGT sequence of integrin β3 cytoplasmic tail in signal transduction by using a dominant negative cell model. Constructs encoding a chimeric protein composed of the extracellular and transmembrane domains of interleukin-2 receptor (Tac) and the full length or RGT-truncated β3 intracellular domain were expressed in CHO cells expressing GPIbIX and integrin αIIbβ3 (IbIX/ IIbIIIa-CHO cell line) to establish the stable IbIX/IIbIIIa-CHO/Tac-β3 and IbIX/IIbIIIa-CHO/Tac-β3Δ759 cell lines. Competitive ELISA was performed to quantify the expression level of Tac-β3 chimeras in the dominant negative cell lines. Spreading and stable adhesion of the IbIX/IIbIIIa-CHO/Tac-β3 and IbIX/IIbIIIa-CHO/Tac-β3Δ759 cells on immobilized fibrinogen (Fg) were examined to evaluate the transduction of outside-in signals. The interaction of Tac-β3 chimeras and endogenous β3 with Src kinase was simultaneously analyzed with Co-IP. Competitive ELISA showed that the expression level of Tac-β3 chimeras was substantially higher than that of endogenous β3 in both IbIX/IIbIIIa-CHO/Tac-β3 and IbIX/IIbIIIa-CHO/Tac-β3Δ759 cell lines that ensures the dominant negative effect of the Tac-β3 chimeras over the endogenous wild type β3 in binding intracellular molecules. In deed， the ability of IbIX/IIbIIIa-CHO/Tac-β3 cells in spreading and stable adhesion on immobilized Fg was effectively inhibited， while that of IbIX/IIbIIIa-CHO/Tac-β3Δ759 cells was not affected. Co-IP results demonstrate that the Tac-β3 chimeras competed with the endogenous integrin β3 in binding Src， while Tac-β3Δ759 lacking the RGT sequence at the C terminal of β3 cytoplasmic tail lost this ability. Selective disruption of Src-binding capacity of Tac-β3 is sufficient to eliminate its dominant negative effect on outside-in signaling suggesting that the interaction of the RGT sequence at the integrin β3 tail with Src kinase is crucial for this signaling pathway.

CSTR: 32200.14.cjcb.2012.02.0005