Reversing thromboxane A2 receptor activity from calcium to cAMP signaling by shifting Gαq to Gαs covalently linked to the receptor

Abstract

The conversion of a G-protein coupling receptor (GPCR) signaling from one G-protein to another could be a novel concept for drug designs. Thromboxane A2 (TXA2) receptor (TP) plays a key role in mediating platelet aggregation and thrombosis through its binding to TXA2 and coupling with Gαq-calcium signaling. To alter TP coupling from Gαq to Gαs, which mediates the opposite anti-platelet aggregation and vasodilation activities, we first created a recombinant single-chain (SC) TP-Gαq complex (SC-TP-Gαq) in which the Gαq N-terminus is covalently linked to the TP C-terminus. The SC-TP-Gαq expressed on HEK293 cells could perform identical functions of wild-type TP binding to agonist and activating Gαq triggering Ca2+ signal. The results were further confirmed by expressing the SC-TP-Gαq on CRISPR-edited Gαq-knocked-out HEK293 cells. A step further, by replacing Gαq using Gαs for SC-TP-Gαq, a second recombinant SC-TP-Gαs was created and expressed on wild type and CRISPR-edited Gαs-knocked-out HEK293 cells. Upon the binding of the same TXA2 agonist to the cells expressing SC-TP-Gαs, cAMP signaling was observed. The results have provided strong evidence that covalently linking TP with different Gα could control cell signaling. This study has opened a door to developing a method to redirect TP signaling from thrombotic calcium to anti-thrombotic cAMP, and apply it to that of other GPCRs.