Essential strategies for the detection of constitutive and ligand-dependent Gi-directed activity of 7TM receptors using bioluminescence resonance energy transfer

The constitutive (ligand-independent) signaling of G protein-coupled receptors (GPCRs) is gaining recognition as a crucial component of their overall function. However, detecting this type of signaling remains technically challenging, especially for poorly characterized receptors, such as orphan receptors in the cAMP-inhibitory Gi-coupled (GiPCR) family. In this study, we explore optimal approaches for detecting constitutive and ligand-modulated activity across several GiPCRs in two different cell lines. As model receptors, we selected two canonical GiPCRs—the constitutively active Smoothened (SMO) and the ligand-activated CXCR4—as well as the atypical GPCR, chemokine receptor ACKR3. We validated the effectiveness of three Bioluminescence Resonance Energy Transfer (BRET)-based assays to assess receptor activity: one that measures changes in intracellular cAMP levels, another that detects Gβγ/GRK3ct association, and a third that monitors Smoothened Agonist Gαi-Gβγ dissociation. These assays were used to analyze both constitutive and ligand-induced signaling of the receptors. In doing so, we identified potential pitfalls and sources of false positives, offering strategies for assay optimization. Our results confirmed the ligand-dependent activation of CXCR4, the G protein incompetence of ACKR3, and the constitutive Gi-directed signaling of SMO, which is modulated by PTCH1. We also discovered that PTCH1 enhances SMO’s cell surface localization, increasing its responsiveness not only to agonists but also to antagonists. This finding reveals a novel regulatory mechanism for the Class F GiPCR Smoothened.