NuPath, a higher-sensitivity TRUPATH-derived biosensor, expands detection of basal and agonist-induced GPCR activation.

Bioluminescence resonance energy transfer (BRET) biosensors allow direct quantification of heterotrimeric G protein activation downstream of G protein-coupled receptors (GPCRs), although restricted dynamic range can limit the detection of subtle, subunit-specific, or constitutively active signalling. We engineered NuPath, a TRUPATH-based biosensor in which Renilla luciferase 8 (RLuc8) within Gα was substituted with NanoLuc luciferase (nLuc) and enhanced yellow fluorescent protein (eYFP) replaced green fluorescent protein 2 (GFP2) fused to Gγ, while preserving the original sensor insertion architecture. NuPath is adapted for use in conventional 96-well plate format and supports direct, no-transfer readouts after receptor expression, whether receptors are introduced transiently or via stable cell lines, thereby simplifying experimental procedures. Side-by-side comparisons of matched NuPath and TRUPATH systems were performed across six GPCRs, Gαi2, GαOA, GαsL, and Gα11, and a range of Gβγ dimers. NuPath produced approximately 3- to 14-fold greater maximal agonist responses with Gαi2, GαsL, and Gα11, whereas GαOA sensors produced responses of broadly comparable magnitude with lower variability. The broadened dynamic range uncovered receptor-dependent variations in Gβγ pairing that were largely undetectable in TRUPATH measurements. NuPath further enabled detection of inverse agonist-mediated suppression of constitutive signalling at the growth hormone secretagogue receptor (GHSR) by liver-expressed antimicrobial peptide 2 (LEAP2), an effect not discernible using TRUPATH. AlphaFold 3 structural models suggested reduced donor-acceptor separation and diminished displacement of the Gα α-helical domain in NuPath compared with equivalent TRUPATH complexes. Collectively, these results position NuPath as a robust, modular enhancement of TRUPATH for resolving agonist-driven, Gβγ-specific, and constitutive GPCR signalling.