Cancer-Associated Mutations Enhance The Sensitivity Of The Trupath GαQ/11 System

ABSTRACT G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are a common drug target. They can be stabilised in different conformational states by ligands to activate multiple transducers and effectors leading to a variety of cellular responses. The potential of agonists to activate select pathways has important implications for drug discovery. Thus, there is a clear need to profile the initial GPCR signal transduction event, activation of G proteins, to enhance understanding of receptor coupling and guide drug design. The BRET-based biosensor suite, TRUPATH, was recently developed to enable quantification of the activation profiles of all non-visual G proteins (excluding G olf and G 14 ) and has since been utilised in numerous studies. However, it fails to detect G q/11 activation for a number of GPCRs previously reported to display promiscuous secondary coupling to G q/11 . Here we report modifications to the Gα q and Gα 11 biosensors in the switch I region that prevent intrinsic GTPase activity (R183C/Q). Except for the PAC1R, substitution with cancer-associated mutations, Cys or Gln, significantly increased sensitivity to allow detection of robust, reliable, and representative G q/11 responses to Class B1 GPCRs. We also demonstrate the utility of these modified biosensors for promiscuously coupled class A GPCR that have primary G s -coupling. Thus, we propose that modification to Gα q/11 may also be necessary in other biosensor systems to enable detection of G q/11 activation.