Critical Residues in the Gβ Subunit Mediate Selective Recruitment of the Gαq Heterotrimer to Activated G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs), the largest family of membrane proteins, serve as primary targets for over 30% of FDA-approved drugs. While signaling bias among Gα subtypes is well characterized, emerging evidence highlights that Gβγ subtypes govern the coupling of the Gα heterotrimer to GPCRs. However, whether Gβγ subtype selectivity depends on the receptor or the Gα subunit and the underlying mechanism remain unresolved. Here, we focused on Gαq-coupled GPCRs and investigated Gβγ preferences using bioluminescence resonance energy transfer assays to assess Gβγ selectivity along with Gαq-GPCR coupling and calcium flux assays to measure downstream Ca²⁺ mobilization. By characterizing seven Gαq-coupled receptors, including the muscarinic acetylcholine receptor family (M1 mAChR, M3 mAChR, M5 mAChR) and four other Gαq-coupled GPCRs (AT1R, NTSR1, KISS1R, hM3Dq), the Gβ1/2γ2 combinations were identified as the preferred subtypes for Gαq coupling. Structural analysis and site-directed mutagenesis assays revealed that three key residues in Gβ2 (F111, V178, and G179), distinct from Gβ4, are crucial for enhancing Gαq coupling. Notably, Gβ4 exhibited preferential recruitment of the Gαq heterotrimer in response to pilocarpine relative to acetylcholine, suggesting that pilocarpine may function as a Gβ4-biased agonist. These findings advance our understanding of Gβγ-mediated subtype selectivity and open avenues for developing biased ligands in drug discovery.