A comprehensive LinkLight platform for GPCR mechanistic profiling to support cancer drug discovery

Background: G protein-coupled receptors (GPCRs) are increasingly recognized as important targets in cancer therapy due to their roles in tumor growth, angiogenesis, and metastasis. LinkLight technology is a protein-protein interaction (PPI) detection platform that monitors beta-arrestin recruitment to activated G protein-coupled receptors (GPCRs), complementing second messenger-based assays for ligand activity characterization. Here, we use LinkLight stable cell lines co-expressing TEV-protease-tagged GPCRs and permuted luciferase (pLuc)-tagged beta-arrestin to study beta-arrestin recruitment, cAMP signal, and calcium-influx within the same cellular context. Methods: Cell-based functional assays were conducted in both agonist and antagonist modes across three GPCR LinkLight stable cell lines representing distinct G-protein couplings (Gs, Gi, and Gq). beta-Arrestin-1/2 recruitment was quantified using the LinkLight PPI luminescence readout, while G-protein-mediated signaling was measured via cAMP or calcium-influx fluorescence, depending on receptor class. Results: Ligands produced consistent, mechanism-appropriate responses across beta-arrestin and second-messenger pathways. For Gq-coupled ADRA1A, the agonist Cirazoline induced robust beta-arrestin-1/2 recruitment (EC₅₀ = 2.51×10⁻⁸ M) and calcium influx (EC₅₀ = 8.2×10⁻⁹ M), while the antagonist Prazosin inhibited both (beta-arrestin EC₅₀ = 2.51×10⁻⁸ M; calcium EC₅₀ = 2.06×10⁻⁷ M). For Gi-coupled ADRA2A, the agonist Brimonidine stimulated beta-arrestin-1/2 recruitment (EC₅₀ = 2.64×10⁻⁸ M) and suppressed cAMP (EC₅₀ = 8.88×10⁻¹⁰ M), whereas the antagonist Yohimbine blocked beta-arrestin recruitment (EC₅₀ = 1.78×10⁻⁸ M) and reversed cAMP inhibition (EC₅₀ = 2.69×10⁻⁷ M). For Gs-coupled ADRB2, the agonist Fenoterol induced beta-arrestin-2 recruitment (EC₅₀ = 8.83×10⁻¹⁰ M) and cAMP signaling (EC₅₀ = 1.52×10⁻⁹ M), while the antagonist Yohimbine (S)-Propranolol inhibited both (beta-arrestin EC₅₀ = 3.98×10⁻⁸ M; cAMP EC₅₀ = 3.6×10⁻⁸ M). Across all receptors, beta-arrestin recruitment aligned with G-protein-specific signaling in response to the same ligand, demonstrating the robustness of the comprehensive GPCR portfolio. Summary: By enabling simultaneous assessment of beta-arrestin recruitment and G-protein signaling in a unified cellular context, LinkLight technology offers a comprehensive, mechanistically informative platform for GPCR pharmacology. Integrated with G-protein activation data, LinkLight technology also supports high throughput profiling of ligand efficacy, bias, and receptor regulation for GPCR drug discovery against cancer.